Heterodimeric osteogenic factor

ABSTRACT

The present invention provides osteogenically active protein preparations comprising a heterodimer of P3 OF 31-34 subunit B and P3 OF 31-34 subunit D, which subunits are linked with at least one disulfide bond and methods for their preparation. The invention further provides cell lines transformed with nucleotide sequences encoding P3 OF 31-34 subunit B and P3 OF 31-34 subunit D and vectors comprising those sequences in operative association with an expression control sequence.

This is a divisional of application Ser. No. 07/718,274, filed Jun. 20,1991, issued Feb. 8, 1994 as U.S. Pat. No. 5,284,756 which is acontinuation-in-part of application Ser. No. 07/415,555, filed Oct. 4,1989 which issued Apr. 21, 1992 as U.S. Pat. No. 5,106,626 and which isa continuation-in-part of application Ser. No. 07/256,034, filed Oct.11, 1988, now abandoned.

BACKGROUND OF THE INVENTION

The present invention relates to novel preparations of osteogenicfactors, methods for their isolation and uses thereof (to repair bonedefects). The preparations so isolated exhibit the ability to promote orstimulate the formation of bone at the site of their application. Boneis a highly specialized connective tissue with unique mechanicalproperties derived from its extensive matrix structure. A network offibrous bundles composed of the protein, collagen, is presumed toprovide the tension-resistant behavior of bone. In addition, othermaterials including proteoglycans, noncollagenous proteins, lipids andacidic proteins associated with a mineral phase consisting primarily ofpoorly crystallized hydroxyapatite are deposited in the extensive matrixarchitecture of bone. Bone tissue is continuously renewed, by a processreferred to as remodeling, throughout the life of mammals. Thisphysiologic process might serve to maintain the properties of a youngtissue.

The processes of bone formation and renewal are carried out byspecialized cells. Osteogenesis vis-a-vis morphogenesis and growth ofbone is presumably carried out by the "osteoblasts" (bone-formingcells). Remodeling of bone is apparently brought about by an interplaybetween the activities of the bone-resorbing cells called "osteoclasts"and the bone-forming osteoblasts. The bony skeleton is thus not only anarchitectural structure with a mechanical function but also is a livingtissue capable of growth, modeling, remodeling and repair. Since theseprocesses are carried out by specialized living cells, chemical(pharmaceutical/hormonal), physical and physicochemical alterations canaffect the quality, quantity and shaping of bone tissue.

A variety of pathological disorders as well as physical stress (forexample, fracture) necessitate active formation of bone tissue at ratesthat are significantly higher than that which can be supported by thenormal milieu of the body. It is thus of value to identifyphysiologically acceptable substances (hormones/pharmaceuticals/growthfactors) that can induce the formation of bone at a predetermined sitewhere such substances are applied, for example, by implantation. Suchagents could either provide a permissive matrix structure for thedeposition of bone-forming cells, or stimulate bone-forming cells, orinduce the differentiation of appropriate progenitors of bone-formingcells.

The presence of proteinaceous and prostaglandin-like growth stimulatorsfor osteoblasts has been examined, see reviews: Raisz, et al., New Engl.J. Med., 309(1), 29-35 (1983) and Raisz, et al., New Engl. J. Med.,309(2), 83-89 (1983).

The observation that a bone graft from the same individual or acompatible individual leads to the formation of new healthy bone at thesite of the graft, led to the hypothesis that bone contains activeproteins which promote local osteogenesis. Urist, et al. disclosedevidence that bone matrix-associated noncollagenous proteins can beisolated by dissociative treatment of demineralized bone powder and thatthis mixture of noncollagenous proteins contain the local osteoinductivecapability which was designated by Urist (e.g., Science, 150, 893(1965)) as bone morphogenetic activity.

A variety of osteogenic, cartilage-inducing and bone-inducing proteinpreparations have been described in the art. Urist, et al. and othershave described various partially fractionated protein preparations withosteoinductive properties. These preparations are fractionated from thenoncollagenous protein mixture extracted using different dissociativetreatment of demineralized bone powder and subjecting the extract tovarious protein fractionation steps. Several such preparations have beencharacterized by different assays to determine their biologicalactivities and by protein components identified using different standardprotein analytical methods.

Urist, et al., Proc. Natl. Acad. Sci. (USA), 81, 371-375 (1984),discloses that bovine BMP has an apparent molecular weight of 18.5Kdaltons. The publication further discloses other bone derived proteinswith apparent molecular weights of 17.5K and 17K, proteins with highermolecular weights of 34K, 24K and 22K and a protein with a lowermolecular weight of 14K. The publication provided the N-terminalsequence for the 17.5K protein which had an unblocked amino terminus.

Urist, European Patent Application No. 212,474, discloses peptidefragments having molecular weights between about 4K and 7K comprising atleast an active portion of the osteoinductive and immunoreactive domainof the 17.5K BMP molecule.

Wang, et al., Patent Cooperation Treaty Application No. WO 88/00205,discloses a bovine bone inductive factor which is isolated fromdemineralized bone powder by a procedure comprising a number ofchromatographic and dialysis steps. The bone inductive factor soisolated was found to contain, as judged by a non-reducing SDS-PAGEanalysis, one or more proteins having a molecular weight ofapproximately 28,000 to 30,000 daltons. Reducing SDS-PAGE analysis ofthe active protein(s) yielded two major bands having the mobility ofproteins having molecular weights of 18,000 daltons and 20,000 daltonsrespectively. Wang, et al., discloses three bovine proteins designatedBMP-1, BMP-2 and BMP-3 where BMP is bone morphogenetic protein andprovides peptide sequences for the proteins. Wang, et al., alsodiscloses the nucleotide sequences and amino acid sequences predictedthereby of four human proteins designated BMP-1, BMP-2 Class I, BMP-2Class II and BMP-3.

Wozney, et al., Science, 242, 1528-1533 (1988), describes the nucleotidesequences and amino acid sequences predicted thereby of three humancomplementary DNA clones (designated BMP-1, BMP-2A and BMP-3)corresponding to three polypeptides present in an extract of bovine bonewhich is capable of inducing de novo bone formation. Recombinant humanBMP-1, BMP-2A and BMP-3 proteins were said to be independently capableof inducing the formation of cartilage in vivo. The nucleotide sequenceand derived amino acid sequence of a fourth complementary DNA clone(designated BMP-2B) is also described. The BMP-1, BMP-2A, BMP-2B andBMP-3 proteins of this publication appear to correspond, respectively,to the BMP-1, BMP-2 Class I, BMP-2 Class II and BMP-3 proteins.

Kubersampath, et al., Patent Cooperation Treaty Application No. WO89/09787 claiming priority based on applications including U.S. Ser. No.179,406 filed Apr. 8, 1988 and Oppermann, et al., Patent CooperationTreaty Application No. WO 89/09788 claiming priority based onapplications including U.S. Ser. No. 179,406 filed April 8, 1988disclose nucleotide sequences and amino acid sequences predicted therebyof a human protein designated OP-1 and certain consensus nucleotidesequences and their amino acid sequences predicted thereby. Theserecombinant proteins are said to be independently capable of inducingthe formation of bone in vivo.

Wang, et al., Proc. Natl. Acad. Sci. USA, 87, pp. 2220-2224 (1990),describe the nucleotide sequence and amino acid sequence predictedthereby of a human protein designated BMP-2A, corresponding to apolypeptide present in an extract of bovine bone which is capable ofinducing de novo bone formation. Recombinant human BMP-2A protein issaid to be independently capable of inducing the formation of bone invivo.

Kubersampath, et al., J. Biol. Chem., 265, 13198-13205 (1990), describesa bovine bone-derived protein that induces bone formation. Thebone-inductive protein was found to contain, as judged by a non-reducingSDS-PAGE analysis, a protein with a molecular weight of approximately30,000 daltons. Reducing SDS-PAGE analysis yielded two major bandscorresponding to molecular weights of 18,000 and 16,000 daltons. The18,000-dalton subunit is the protein product of the bovine equivalent ofthe human OP-1 gene and the 16,000-dalton subunit is the protein productof the bovine equivalent of the human BMP-2A gene.

Celeste, et al., Proc. Natl. Acad. Sci. USA, 87, 9843-9847 (1990),describe the human protein sequences derived from the nucleotidesequence of six genes encoding proteins related to TGF-β. These encodedproteins are designated BMP-2, BMP-3, BMP-4, BMP-5, BMP-6 and BMP-7.

SUMMARY OF THE INVENTION

The present invention is directed to novel preparations of osteogenicfactors, methods for their isolation and uses thereof. Specifically, theinvention is based on the discovery that a primary osteogenically activeprotein of P3 OF 31-34 is a heterodimer of P3 OF 31-34 subunit B(hereinafter "subunit B") and P3 OF 31-34 subunit D (hereinafter"subunit D"). Preparations comprising the B/D heterodimer arecharacterized by the ability to stimulate osteogenesis. The inventionprovides a method of producing an osteogenic protein preparationcomprising a heterodimer of a first polypeptide subunit and a secondpolypeptide subunit comprising the steps of culturing in a suitableculture media one or more cell lines transformed with a first and asecond nucleotide sequence, said first nucleotide sequence beingselected from the group consisting of: the nucleotide sequence encodingsubunit B as shown in SEQ ID NO: 3; a nucleotide sequence which encodesthe same sequence of amino acids as encoded by the nucleotide sequenceshown in SEQ ID NO: 3; a nucleotide sequence which is at least 80%homologous with the nucleotide sequence shown in SEQ ID NO: 3 and whichencodes a homologue of subunit B having the osteogenic activity of P3 OF31-34 subunit B; and a nucleotide sequence which would be at least 80%homologous with the nucleotide sequence shown in SEQ ID NO: 3 but forthe redundancy of the genetic code and which encodes a homologue ofsubunit B having the osteogenic activity of P3 OF 31-34 subunit B. Thesecond nucleotide sequence is selected from the group consisting of: thenucleotide sequence encoding subunit D as shown in SEQ ID NO: 1; anucleotide sequence which encodes the same sequence of amino acids asencoded by the nucleotide sequence shown in SEQ ID NO: 1; a nucleotidesequence which is at least 80% homologous with the nucleotide sequenceshown in SEQ ID NO: 1 and which encodes a homologue of subunit D havingthe osteogenic activity of P3 OF 31-34 subunit D; and a nucleotidesequence which would be at least 80% homologous with the nucleotidesequence shown in SEQ ID NO: 1 but for the redundancy of the geneticcode and which encodes a homologue of subunit D having the osteogenicactivity of P3 OF 31-34 subunit D. The cell line(s) is (are) cultured toproduce the first and second polypeptide subunits which are linked witha disulfide bond to form heterodimers and are isolated. The B/Dheterodimers are preferably purified and isolated according to the stepsof subjecting the culture medium to a series of chromatography stepsutilizing a Q-Sepharose column, an S-Sepharose column and aPhenyl-Sepharose column to recover an active fraction. The activefraction is then subjected to reverse phase chromatography using a C-18high performance liquid chromatography column equilibrated with bufferscontaining trifluoroacetic acid and acetonitrile by eluting the activepreparation at concentrations between 35% and 45% acetonitrile. Theinvention further provides the osteogenic preparations prepared therebyand pharmaceutical products comprising the osteogenic preparation somade. Also provided by the invention is a method for transforming a cellwith genes encoding both subunits B and D and homologues thereof andcells transformed thereby. The invention further provides vectorscomprising a first and a second DNA sequence in operative associationwith an expression control sequence which sequence encode subunits B andD or homologies thereof. In addition, the invention provides a methodfor inducing bone formation in a mammal comprising administering to themammal an effective amount of the osteogenic preparation comprisingheterodimers of subunits B and D or homologues thereof. The inventionfurther provides compositions for implantation into a mammal comprisingthe osteogenic preparation comprising heterodimers of subunits B and Dadmixed with a physiologically acceptable matrix material.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A and 1B illustrate a method for the purification of P3 OF 31-34(osteogenic factors) proteins from calf bone.

FIG. 2A shows the apparent molecular weight of the osteogenic factors asdetermined by non-reducing SDS polyacrylamide gel electrophoresisfollowed by silver staining.

FIG. 2B shows reducing SDS polyacrylamide gel electrophoresis of P3 OF31-34 proteins followed by silver staining.

FIG. 3A shows the isolation of the subunits of the P3 OF 31-34 proteins(osteogenic factors) by reverse phase HPLC.

FIG. 3B shows the apparent molecular weights of the subunits as detectedby silver staining of reducing SDS polyacrylamide gel electrophoreticanalysis.

FIG. 4A represents the elution profile obtained by high performanceliquid chromatography, on a reverse phase C18 column, of the PS Pool.

FIG. 4B shows non-reducing SDS polyacrylamide gel electrophoresis of P3OF 31-34 proteins eluting in fractions 26, 27 and 28 from the reversephase HPLC of the PS Pool.

FIG. 5A shows the isolation and identification of subunits of the P3 OF31-34 proteins eluting in fraction 26, from the reverse phase HPLC ofthe PS Pool.

FIG. 5B shows the isolation and identification of subunits of the P3 OF31-34 proteins eluting in fraction 28 from the reverse phase HPLC of thePS Pool.

FIG. 6 shows the nucleotide and derived amino acid sequences, also setout in SEQ ID NOS: 1 and 2, of the cDNA gene for human mature D.

FIG. 7 shows the nucleotide and derived amino acid sequences, also setout in SEQ ID NOS: 3 and 4, Of the/cDNA gene for human mature B.

FIGS. 8A, 8B and 8C shows the nucleotide and derived amino acidsequences, also set out in SEQ ID NOS: 5 and 6, of the cDNA gene forhuman C.

FIG. 9 shows reducing and non-reducing SDS polyacrylamide gelelectrophoresis of enriched samples isolated from media of CHO cellstransfected with both the gene sequence for human mature B and the genesequence for human mature D (Prep B/D), or separately transfected withonly the gene sequence for human mature D (Prep D), or with only thegene sequence for human mature B (Prep B). Proteins were visualizedusing autoradiography following Western Blot analysis.

FIG. 10 shows the biological activity of the enriched samples of PrepB/D, Prep B+D, Prep D and Prep B as determined using the rat implantassay following implantation of various amounts of immunoreactivity.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to the osteogenic preparation known as P3OF 31-34 which has previously been characterized by applicants ascomprising osteogenically active proteins which during gel filtrationduring non-reducing and dissociative conditions, elute as proteinshaving apparent molecular weights within the range of about 31,000 to34,000 daltons. In co-owned and copending U.S. patent Application Ser.No. 07/415,555, filed Oct. 4, 1989 the disclosure of which is herebyincorporated by reference it is disclosed that the P3 OF 31-34osteogenic protein material yields four distinct peaks when analyzed byreverse phase HPLC after reduction. When analyzed by reducing SDS-PAGEand silver staining, three of the peaks are characterized as proteinsubunits migrating with apparent molecular weights within the range of17,500 to 19,000 daltons, and the fourth peak is characterized as aprotein subunit migrating with an apparent molecular weight within therange of 16,000 to 17,500 daltons. The polypeptide subunits of P3 OF31-34 have been designated as subunits A, B, C and D. The subunits havebeen characterized by amino acid sequences and cDNA sequences encodingthe subunits. The present invention is based on the discovery that aprimary osteogenically active protein comprises a heterodimer of P3 OF31-34 subunit B and P3 OF 31-34 subunit D wherein the subunits arelinked by at least one disulfide bond.

The osteogenic protein preparation comprising the B/D heterodimer may beused to form a composition for implantation into a mammal by admixturewith a physiologically acceptable matrix material. In addition, devicesfor implantation into mammals comprising a structural member encoatedwith the osteogenic factor/matrix composition are provided by theinvention.

The present invention is intended to encompass osteogenically activeheterodimers of subunit B and D analogues. Specifically, it iscontemplated that various deletions, insertions and substitutions can bemade in the amino acid sequences of subunits B and D such that thesequences will vary from those which are present in naturally derivedmammalian B/D heterodimer. The B/D heterodimer and its subunits can alsobe chemically or enzymatically modified, can be fusion proteins or canbe bound to suitable carrier substances such as a polymer. To the extentthat such molecules retain osteogenic activity, they are contemplated asbeing within the scope of the present invention.

The subunit B and D polypeptides can be produced by expression of DNAprepared by molecular cloning technologies or by chemical synthesis ofoligonucleotide and assembly of the oligonucleotide by any of a numberof techniques prior to expression in a host cell. [See, e.g., Caruthers,U.S. Pat. No. 4,500,707; Balland, et al., Biochimie, 67, 725-736 (1985);Edge, et al., Nature, 292, 756-762 (1981)]. Messenger RNA encodingsubunits B or D or analogs thereof may also be expressed in vitro.Changes in activity levels are measured by the appropriate assay.Modifications of such protein properties as redox or thermal stability,hydrophobicity, susceptibility to proteolytic degradation, or thetendency to aggregate with carriers or into multimers are assayed bymethods well known to those of ordinary skill in the art.

Prokaryotic microorganisms (such as bacteria) and eukaryoticmicroorganisms (such as yeast) may be employed as host cells accordingto the present invention. S. cerevisiae, or common baker's yeast, is themost commonly used among eukaryotic microorganisms, although a number ofother strains are commonly available. For expression in bacteria andyeast, cloning and expression vectors are well known to those skilled inthe art, such as lambda phage and pBR322 in E. coli and YRp7 in S.cerevisiae.

Cells derived from multicellular eukaryotes may also be used as hosts.Cells from vertebrate or invertebrate eukaryotes may be used, and thoseskilled in the art know of appropriate expression vectors for usetherein, such as SV40 retroviral and papilloma viral vectors formammalian host cells, NPV vectors for invertebrate host cells and Tivectors for plant cells.

It is preferred that host cells be transformed with genes encoding bothsubunits B and D in order that intracellular processing link thesubunits with at least one disulfide bond. Nevertheless, it iscontemplated that the subunits can be separately expressed and thesubunits be dimerized in vitro utilizing denaturation/renaturationtechniques known in the art.

The present invention further discloses methods of using the B/Dheterodimers and compositions which comprise them as pharmaceuticalagents for the stimulation of bone growth in mammals. Pharmaceuticallyacceptable compositions comprised of one or more of the proteins and/oractive polypeptides and/or immunologically related entities incombination with a pharmaceutically acceptable carrier are alsodisclosed herein. Such compositions can optionally contain otherbioactive materials or other ingredients which aid in the administrationof the composition or add to the effectiveness of the composition.

The term "osteogenesis" means formation of new bone or induction ofgrowth of pre-existing bones at specific sites in response to localadministration (for example, implantation of an active preparation in apharmaceutically acceptable manner). The term "osteogenic amount" refersto an amount of the osteogenic protein and/or active polypeptide and/orimmunologically related entity sufficient to provide the desired effect.The term "osteogenically active" or "osteogenic" means that thepreparation has the capability to promote or induce osteogenesis.

The application of the osteogenic factors can be convenientlyaccomplished by administering, such as by implanting, a lyophilizedpreparation or suspension of one or more of the osteogenic proteinsand/or one or more active polypeptide and/or one or more immunologicallyrelated entities in sufficient quantity to promote osteogenesis at thedesired site. Alternatively, pharmaceutically acceptable compositionscan be used which are comprised of one or more of the osteogenicproteins and/or one or more of the active polypeptides and/or one ormore of the immunologically related entities described herein and apharmaceutically acceptable matrix such as collagenous proteins ormatrix material derived from powdered bone extracted with strongdenaturing agents, or other pharmaceutically acceptable carriers.

While the B/D heterodimer is a major osteogenically active protein, itis contemplated that preparations comprising the B/D heterodimer incombination with other homo- and heterodimers of P3 0F 31-34 subunits A,B, C, D and E may provide synergistic effects with respect to osteogenicactivity.

The following examples are included to further illustrate the inventionbut are not to be construed as limitations thereon.

EXAMPLE 1 Purification of Bovine Osteogenic Factors

According to this example, bovine osteogenic factors were isolated fromdemineralized calf bone powder according to the procedure disclosed inFIGS. 1A and 1B. Approximately 200 pounds of diaphysial sections of calfbone were scraped clean of connective tissue and marrow was removed. Thedemarrowed sections were ground to a powder and washed withapproximately 2100 liters of cold deionized water. The bone powder wasallowed to settle during the water washes and the suspended connectivetissue fragments were removed with the supernatant and discarded.

The bone powder was suspended in a total of approximately 570 liters ofcold 0.5M HCl for about 2 hours and was then allowed to settle. The HClwas removed with the supernatant and discarded. The remaining HCl wasremoved by washing the bone powder with approximately 700 liters of colddeionized water, followed by approximately 350 liters of cold 0.1M Tris,pH 7, solution. The demineralized bone powder (demineralized bone) wasallowed to settle and the supernatant was discarded.

The demineralized bone powder was suspended in approximately 140 litersof cold 4M guanidine hydrochloride containing 0.01M Tris, pH 7, and0.001M EDTA for about 20 hours. The extracted bone powder was removed byfiltration and discarded. The supernatant (guanidine extract) was saved.

The guanidine extract was filtered through Amicon hollow fibercartridges (H10-P100-20) with an average molecular weight cutoff of100,000 daltons. The 100,000 dalton filtrate (100K filtrate) was thenconcentrated through Amicon spiral cartridges (S10Y10) with molecularweight cutoffs of 10,000 daltons. The 10,000 dalton retentate (10Kretentate) was saved and assayed for pH, conductivity, total proteincontent by BCA colorimetric protein assay (Pierce Chemicals, Rockford,Ill.), resolution of protein constituents in the preparations usingreducing SDS-PAGE followed by silver staining or Coomassie Blue stainingand determination of the osteogenic activity using the rat implant assaydisclosed below in Example 2.

The 10K retentate was exchanged into 6M urea containing 50 mM2-(N-morpholino)ethanesulfonic acid (MES), pH 6.5, by diafiltration withan Amicon spiral cartridge (S10Y10) with a molecular weight cutoff of10,000 daltons.

The diafiltered extract was adjusted to a pH of 6.5 using 5M NaOH and aconductivity of 10 mS/cm using 5M NaCl and applied to a 0.4 literS-Sepharose column (Pharmacia Chemicals, New Jersey) equilibrated with6M urea containing 50 mM MES, pH 6.5, adjusted to conductivity of 10mS/cm. The column was washed with 2.4 liters of 6.0M urea containing 50mM MES, pH 6.5, adjusted to a conductivity of 10 mS/cm to elute theunbound proteins. The S-Sepharose active pool (SS Pool) was eluted with1.2 liters of 6.0M urea containing 50 mM MES, pH 6.5, and 0.5M NaCl. TheS-Sepharose active pool was concentrated using membrane filters with anaverage molecular weight cutoff of 10,000 daltons. The pH andconductivity of the preparation were determined, the total proteincontent was measured by BCA protein assay, the protein constituents wereanalyzed using SDS-PAGE followed by silver staining and the osteogenicactivity was determined using the rat implant assay.

The S-Sepharose active pool was exchanged into 6M urea containing 20 mMethanolamine, pH 9.5 by diafiltration with an Amicon spiral cartridge(S10Y10) with a molecular weight cutoff of 10,000 daltons.

The G-25 Pool was applied to a 0.7 liter Q-Sepharose column (PharmaciaChemicals, New Jersey) equilibrated with 6M urea containing 20 mMethanolamine, pH 9.5. The column was washed with 2.1 liters of 6M ureacontaining 20 mM ethanolamine, pH 9.5, to elute the unbound proteins.The osteogenically active protein pool (QS Pool) was eluted fromQ-Sepharose column with 1.4 liters of 6M urea containing 20 mMethanolamine, pH 9.5, and 0.2M NaCl. The QS Pool was adjusted to a pH of6-7 with glacial acetic acid and concentrated using membrane filterswith an approximate molecular weight cutoff of 10,000 daltons. The QSPool was assayed for pH and conductivity; the total protein content wasdetermined by BCA protein assay, the protein constituents were analyzedby reducing SDS-PAGE followed by silver staining and the osteogenicactivity was measured using the rat implant assay.

The QS Pool was then applied to a preparative C-18 HPLC columnequilibrated with a buffer containing, by volume, 70% Buffer A (Buffer Ais 0.05% trifluoroacetic acid in water) and 30% Buffer B (Buffer B is0.025% trifluoroacetic acid in acetonitrile). Bound proteins were elutedusing a linear gradient of 30% to 60% acetonitrile in 120 minutes. Theosteogenic activity (Prep HPLC Pool) eluted within the concentrations of35% to 45% acetonitrile. The Prep HPLC Pool was lyophilized andresuspended in 1 ml of water. The Prep HPLC Pool was assayed for pH andconductivity; the total protein content was determined by BCA proteinassay, the protein constituents were analyzed by reducing SDS-PAGEfollowed by silver staining and the osteogenic activity was measuredusing the rat implant assay.

The Prep HPLC Pool was adjusted to a protein concentration of 0.5 mg/mlin 6M urea containing 50 mM Tris, pH 7.5-8.0, 20 mM ethanolamine and0.5M NaCl and was applied to a 5-10 ml Chelating-Sepharose 6B column(Pharmacia Chemicals, New Jersey) charged with Cu²⁺ and equilibratedwith 6M urea containing 50 mM Tris, pH 7.5-8.0, 20 mM ethanolamine and0.5M NaCl. The column was washed with 5 column volumes of equilibrationbuffer followed by 10 column volumes of 6 M urea containing 50 mM Tris,pH 7.4-7.8, to elute the unbound proteins. Bound proteins were elutedwith 10 column volumes of 6M urea containing 50 mM Tris, pH 7.4-7.8, and4 mM imidazole. The osteogenic activity (CC Pool) was eluted from thecopper chelate column with 10 column volumes of 6M urea containing 50 mMTris, pH 7.4-7.8, and 15 mM imidazole. The CC Pool was assayed for totalprotein as estimated by absorbance at 280 nm, and its osteogenicactivity was measured using the rat implant assay.

The CC Pool was adjusted to 25% ammonium sulfate and loaded onto a 1-3ml column of Phenyl-Sepharose (Pharmacia Chemicals, New Jersey)equilibrated with 6M urea containing 25% ammonium sulfate, 50 mM Tris pH7.4-7.8. The column was washed with 10 column volumes of 6M ureacontaining 25% ammonium sulfate, and 50 mM Tris pH 7.4-7.8, to elute theunbound proteins. Bound proteins were eluted with 10 column volumes of6M urea containing 15% ammonium sulfate, 50 mM Tris pH 7.4-7.8. Theosteogenic activity (PS Pool) was eluted from the Phenyl-Sepharosecolumn with 6M urea containing 50 mM Tris pH 7.4-7.8, was assayed fortotal protein as estimated by absorbance at 280 nm, and its osteogenicactivity was measured using the rat implant assay.

The PS Pool was applied to a semi-preparative or analytical C-18 HPLCcolumn equilibrated with a buffer containing, by volume, 70% Buffer Aand 30% Buffer B (Buffer A is 0.05% trifluoroacetic acid in water andBuffer B is 0.025% trifluoroacetic acid in acetonitrile). Bound proteinswere eluted using a linear gradient of 30% to 60% acetonitrile. As waspreviously characterized, the osteogenic activity (HPLC Pool) elutedwithin the concentrations of 35% to 45% acetonitrile. The HPLC Pool wasassayed for total protein as estimated by absorbance at 229 nm and itsosteogenic activity was measured using the rat implant assay.

EXAMPLE 2 Biological Activity

The induction of bone matrix was measured using a rat implant assay asgenerally described by Sen, Walker and Einarson (1986), in Developmentand Diseases of Cartilage and Bone Matrix, eds. A. Sen and T. Thornhill,201-220, Alan R. Liss, New York; and Sampath, et al., Proc. Natl. Acad.Sci. (USA), 80, 6591-6595 (1983). Approximately 70-100 mg of inactivebone matrix (bone collagen) was mixed with an aqueous solution ofosteogenic protein preparation and the water removed by lyophilization.The dried coated granules were packed in gelatin capsules (Eli Lilly #5)and each capsule was subcutaneously implanted near the thigh muscles ineach back leg of male Long Evans rats. The implanted rats weresacrificed 17 to 28 days following implantation and the implant tissuewas surgically removed and placed in Bouin's Solution. The specimenswere then decalcified and processed for toluidine blue stained sections.Histomorphology and percent ossification was determined by examinationof the stained sections. Potency is defined by the amount of protein(mg) required for implantation with inactive bone matrix yielding atleast 10% of the area of the stained sections occupied by osteoidactivity.

                  TABLE 1                                                         ______________________________________                                        PURIFICATION OF OSTEOGENIC FACTORS                                                                       Potency in Rat                                     Sample       Total Protein (mg/implant)                                       ______________________________________                                        Guanidine Extract                                                                          130,000-170,000 mg                                               10K Retentate                                                                               6,000-15,000 mg                                                                            10.0                                               S-S Pool     300-900 mg    1.0                                                QS Pool       70-250 mg    0.25                                               Prep HPLC Pool                                                                              4-12 mg      0.05                                               CC Pool      2-5 mg        0.025                                              PS Pool      0.5-1 mg      0.01                                               HPLC Pool    0.01-0.05 mg  0.001                                              ______________________________________                                    

The increase in potency of the various osteogenically active proteinpreparations obtained using purification steps according to Example 1 isshown in Table 1, above, with the HPLC Pool having a potency of 0.001mg/implant.

EXAMPLE 3 Determination of Molecular Weights of Purified OsteogenicFactors Under Reducing and Nonreducing Conditions and Purification ofReduced Subunits

Purified osteogenically active protein preparation as obtained in theHPLC Pool of Example 1 were suspended in SDS dilution buffer in theabsence of reducing reagents (-DTT), electrophoresed on 12.5% or 15% SDSpolyacrylamide gels and the protein bands visualized by silver staining.Molecular weights are determined relative to non-prestained molecularweight standards (Bio-Rad). This gel system revealed that the HPLC Poolcontained protein bands which migrate within the molecular weight rangeof 31,000-34,000 daltons (see FIG. 2A).

Purified osteogenically active proteins in the HPLC Pool were subjectedto an alternative analytical method whereby protein subunits heldtogether by disulfide bonds can be resolved by reduction of these bondsin SDS dilution buffer in the presence of a reducing agent(dithiothreitol or betamecaptoethanol) and electrophoresis on 12.5% or15% SDS polyacrylamide gels. Molecular weights were determined relativeto non-prestained molecular weight standards (Bio-Rad). In this gelsystem, the HPLC Pool revealed proteins migrating as two broad bandswithin the molecular weight ranges of 16,000-17,500 and 17,500-19,000daltons (see FIG. 2B).

The HPLC pool was made 6M in guanidine hydrochloride, 50 mM inethanolamine and 50 mM in dithiothreitol to reduce the disulfide bonds.The reduced sample was diluted at least 2 fold with either water or0.05% trifluoroacetic acid in water and loaded onto an analytical C-18HPLC column equilibrated with a buffer comprising, by volume, 70% BufferA and 30% Buffer B, as described previously (Buffer A is 0.05%trifluoroacetic acid in water and Buffer B is 0.025% trifluoroaceticacid in acetonitrile). Bound proteins were eluted using a lineargradient of 30% to 60% acetonitrile in 60 minutes. Four prominent peaksof protein, designated A, B, C and D, were detected by monitoring UVabsorbance at 229 nm; these eluted within the concentrations of 40% to47% acetonitrile (see FIG. 3A). When analyzed by reducing SDS gelelectrophoresis followed by silver staining, the reduced subunit Amigrated within the molecular weight range of 17,500-19,000 daltons, thereduced subunit B migrated within the molecular weight range of16,000-17,500, the reduced subunit C migrated within the molecularweight range of 17,500-19,000 and the reduced subunit D migrated withinthe molecular weight range of 17,500-19,000 (see FIG. 3B).

EXAMPLE 4 Amino Acid Sequences of Bovine Osteogenically Active ProteinsP3 OF 31-34

The isolated reduced subunits purified from HPLC Pool as disclosed inExample 3, were analyzed by a gas phase sequenator (Applied Biosystems,Model 470A), and found to have the following amino-terminal sequences:

SEQ ID NO: 7

Subunit A: SAPGRRRQQARNRSTPAQDV

SEQ ID NO: 8

Subunit C: SXKHXXQRXRKKNNN

SEQ ID NO: 9

Subunit D: STGGKQRSQNRSKTPKNQEA

where the amino acids are represented by the well known one-letter andthree-letter designations presented in Table 2 below.

                  TABLE 2                                                         ______________________________________                                                       Three-Letter                                                                             One-Letter                                          Amino Acid     Abbreviation                                                                             Symbol                                              ______________________________________                                        Alanine        Ala        A                                                   Arginine       Arg        R                                                   Asparagine     Asn        N                                                   Aspartic Acid  Asp        D                                                   Cysteine       Cys        C                                                   Glutamine      Gln        Q                                                   Glutamic acid  Glu        E                                                   Glycine        Gly        G                                                   Histidine      His        H                                                   Isoleucine     Ile        I                                                   Leucine        Leu        L                                                   Lysine         Lys        K                                                   Methionine     Met        M                                                   Phenylalanine  Phe        F                                                   Proline        Pro        P                                                   Serine         Ser        S                                                   Threonine      Thr        T                                                   Tryptophan     Trp        W                                                   Tyrosine       Tyr        Y                                                   Valine         Val        V                                                   Undetermined              X                                                   ______________________________________                                    

The isolated subunit B yielded no detectable amino-terminal sequence.When subunit B was digested with Staph V8 protease, andrechromatographed by HPLC, two detectable internal fragments wereisolated having the following amino acid sequences:

SEQ ID NO: 10

Subunit B/Staph V8: XVVLKNYQDMV

SEQ ID NO: 11

Subunit B/Staph V8: XXKVVLKNYQDM

where X represents an unassigned amino acid.

The isolated, reduced subunits purified from HPLC Pool (Example 3) wereadsorbed onto polyvinylidine difluoride (PVDF) transfer membrane(Millipore, Bedford, Mass.), exposed to vapors from 80 mg/ml CNBr in 70%formic acid for 15 to 20 hours and sequenced using the gas phasesequenator. The following amino acid sequences are represented by thewell-known one-letter designations presented in Table 2.

Subunit A, following cleavage with CNBr, yielded sequences from thesimultaneous sequences of several fragments corresponding to the aminoterminal sequence:

SEQ ID NO: 12

ANt: SAPGRRRQQARNRSTPAQDV

and three internal fragments:

SEQ ID NO: 13

A1: NPEYVPKXXXAPTKLNAISV

SEQ ID NO: 14

A2: XATNXAIVQXLVXLM

SEQ ID NO: 15

A3: XVXAXG

Subunit B, following cleavage with CNBr, yielded sequences from thesimultaneous sequencing of two internal fragments:

SEQ ID NO: 16

B1: LYLDENEK

SEQ ID NO: 17

B2: VVEGXGXR

when compared with the sequences of fragments of subunit B cleaved withstaph V8 protease, fragments B1 and B2 contain overlapping regions,allowing an extended internal sequence in subunit B:

B1: LYLDENEK (SEQ ID NO: 16)

Staph V8: XXKVVLKNYQDM (SEQ ID NO: 11)

Staph V8: XVVLKNYQDMV (SEQ ID NO: 10)

B2: VVEGXGXR (SEQ ID NO: 17)

Consensus: LYLDENEKVVLKNYQDMVVEGXGXR (SEQ ID NO: 18)

Subunit D, following cleavage with CNBr, yielded sequences from thesimultaneous sequencing of several fragments corresponding to the aminoterminal sequence:

SEQ ID NO: 19

DNt: STGGKQRSQNRSKTPKNQEA

and an internal sequence:

SEQ ID NO: 20

D1: XATNHAIVQTLVHFINXETV

The isolated reduced subunit C, purified from the HPLC Pool (Example 3),was adsorbed onto a PVDF transfer membrane, subjected to 20 cycles ofamino terminal sequencing using the gas phase sequenator, subjected tocleavage by CNBr vapors, and then sequenced using the gas phasesequenator. Subunit C, following cleavage with CNBr, yielded thefollowing internal sequences:

SEQ ID NO: 21

C1: LYLXEYDXVVLXNYQ

SEQ ID NO: 22

C2: SAXXHXIVQT

The amino terminal and internal sequences of subunits A, B, C and Dderived from bovine bone can be aligned with homologous regions from thededuced amino acid sequences of cDNA clones encoding the polypeptidesdesignated BMP-2A, BMP-2B and Vgr-1. (Wozney, et al., Science, Vol. 242,pp. 1528-1534 (1988) and (Lyons, et al., Proceedings of the NationalAcademy of Sciences of the U.S.A., Vol. 86, pp. 4554-4558, 1989).Comparison of the similarities and differences of the sequences ofsubunits B and C and the sequences of BMP-2A and BMP-2B indicate thatbovine subunit B shares the same sequence as BMP-2A while bovine subunitC shares the same sequence as BMP-2B. The amino terminus of mature B isinferred from alignment of the human BMP-2A sequence with the amino acidsequences of bovine A, B, C and D, and the presence of a blocked aminoterminal on bovine subunit B as described above, presumably resultingfrom cyclization of an N-terminal glutamine to form a nonsequenceablepyroglutamic acid. Alignment of the sequences of subunit A with those ofVgr-1 indicates a 90% homology.

EXAMPLE 5 Subunit Compositions of Purified Osteogenically ActiveProteins p3 OF 31-34

Individual fractions, eluting within the HPLC pool (Example 1) andcontaining the osteogenically active proteins P3 OF 31-34 (FIG. 4A),wereanalyzed by SDS polyacrylamide gel electrophoresis in the absence ofreducing reagents (FIG. 4B). FIG. 4A shows the elution profile obtainedby high performance liquid chromatography, on a reverse phase C18 columnof the PS Pool. FIG. 4B shows non-reducing SDS polyacrylamide gelelectrophoresis of P3 OF 31-34 proteins eluting in fractions 26, 27 and28 from the reverse phase HPLC of the PS Pool. These individualfractions were further analyzed (as described in Example 3) by reductionof the disulfide bonds with 50 mM dithiothreitol in 50 mM ethanolamineand 6M guanidine hydrochloride and chromatography on a C18 HPLC column(FIG. 5). FIG. 5A shows the isolation and identification of subunits ofthe P3 of 31-34 proteins eluting in fraction 26 from the reverse phaseHPLC of the PS Pool, while FIG. 5B shows the isolation andidentification of P3 OF 31-34 proteins eluting in fraction 28. subunitsA, B, C and D are designated by the solid lines in the figures. Fraction26, the sample comprising the lowermost band of the P3 OF 31-34 region(Band I of FIG. 4B), was found to contain predominantly subunits B and Dwith smaller amounts of subunits A and C. Fraction 28, the samplecomprising predominantly the uppermost band of the P3 OF 31-34 region(Band II of FIG. 4B), together with a small amount of Band I, was foundto contain increased amounts of subunits A and C, and a decreased amountof subunit D.

These individual fractions, eluting within the HPLC pool and containingthe osteogenically active proteins P3 of 31-34, were electrophoresed on12.5% SDS polyacrylamide gels in the absence of reducing reagent (-DTT),electrophoretically transferred to polyvinylidine difluoride (PVDF)transfer membranes in the presence of 10% methanol, 10 mMcyclohexylamino-1-propanesulfonic acid, pH 10-11, at 0.5 amp for 15 to30 minutes, and visualized by staining with Coomassie Brilliant BlueR250. Individual protein bands in the region of P3 OF 31-34 defined hereas Band I (lower) and Band II (upper), were sliced from the membrane andsubjected first to N-terminal sequencing, and then to internalsequencing following treatment with CNBr as described in Example 4.These procedures revealed the following sequence for Band I and II:

    ______________________________________                                                                       Subunit                                        Band Sequenced Sequences       Identity                                       ______________________________________                                        Band I  Internal   XATNXAIVQTL     D                                                             (SEQ ID NO: 23)                                                               LYLDEXEXVVL     B                                                             (SEQ ID NO: 24)                                            Band II N-Terminal XXXGRXRQ        A                                                             (SEQ ID NO: 25)                                                               XXGGXQR         D                                                             (SEQ ID NO: 26)                                            Band II Internal   LYLDXNXXVVLXN   B                                                             (SEQ ID NO: 27)                                                               XPEXVPX         A                                                             (SEQ ID NO: 28)                                            ______________________________________                                    

where the amino acids are represented by the well-known one-letterdesignations presented in Table 2.

These results indicated that Band I, the lowermost band of the P3 OF31-34 proteins, contains predominantly subunits D and B, and that BandII, the uppermost band of the P3 of 31-34 proteins, containspredominantly subunits A and B. These compositions, as well as theobservation that these subunits are purified as disulfide-linked dimersin the purified P3 OF 31-34 proteins (Example 3), indicate that subunitsD and B may be disulfide-linked as a heterodimer, and that subunits Aand B may be disulfide-linked as another heterodimer.

EXAMPLE 6 Osteogenic Compositions for Implantation

The osteogenic preparations of the invention may be used to prepareosteogenic compositions for implantation into mammals. The osteogenicprotein may be admixed with one or more of a variety of physiologicallyacceptable matrices. Such matrices may be resorbable, non-resorbable orpartially resorbable. Resorbable matrices include polylactic acidpolycaprolactic acid, polyglycolic acid, collagen, plaster of paris anda variety of thermoplastic polymer materials. Non-resorbable materialsinclude hydroxyapatite and partially resorbable materials includematrices such as tricalcium phosphate. The osteogenic protein may beadsorbed onto the matrix material which can be either in a granular orsolid form. The osteogenic composition may then be dried bylyophilization.

EXAMPLE 7 Device Coated With Osteogenic preparations

In this example, the Prep HPLC Pool of Example 1 containing theosteogenically active proteins was used to form osteogenically activedevices useful for the healing of bone defects. The devices wereprepared by absorbing the Prep HPLC Pool onto solid delivery matricescomprising either a porous hydroxyapatite disc (Interpore 200, InterporeInternational, Irvine, Calif.) or a porous polylactic acid disc (DRILAC,OSMED Incorporated, Costa Mesa, Calif.). The discs were 8 to 10 mm indiameter and 3 mm thick and were coated with 0.2 to 0.3 mg of the PrepHPLC Pool which was dried onto the matrix by lyophilization. The devicemay then be sterilized by gamma-irradiation with as much as 3.3 to 3.5Mrads or other suitable means. The devices comprising the osteogenicpreparation and the matrix were implanted into trephine defects createdin New Zealand Albino Female rabbits, weighing 2.5 to 3.0 kg.Specifically, test devices either coated with the osteogenic preparationor not coated with the osteogenic preparation were surgically implantedinto the calvaria using appropriate aseptic surgical techniques. Animalswere anesthetized with an intramuscular injection of Ketamine andXylazine. Following a midline incision, the calvarium was exposed andtwo trephine holes (one on each side of the midline) 5 mm posterior tothe orbits, 8-10 mm in diameter and to the depth of the dura were cutinto the calvarium. Trephine defects were created using a Stille cranialdrill, exercising great care not to injure the dura. A test device wasimplanted into one trephine hole while the trephine hole on the oppositeside was left empty. Following surgical implantation, antibioticprophylaxis with penicillin and streptomycin was administered. Theanimals were followed daily by clinical observations. At explant, thecalvaria was removed en block. The specimens were fixed in 10% bufferedformalin, decalcified and processed for hematoxylin and eosin stainedsections. Histomorphology and qualitative determination of percentossification was determined by examination of the stained sections. (seeTable 3 below). The percent area of activity is estimated by eye fromthe fields of view, or fraction of fields of view, of newly formed bonematrix as compared to the total fields of view not occupied by thematrix in the entire full cross section.

                  TABLE 3                                                         ______________________________________                                        % OSSIFICATION IN DEVICES                                                     IMPLANTED INTO RABBIT TREPHINE DEFECTS                                                         Time of Explant                                              Test Device        6 weeks  12 weeks                                          ______________________________________                                        Uncoated Hydroxyapatite                                                                          <10%     <25%                                              Uncoated Polylactic Acid                                                                         <10%     <10%                                              Hydroxyapatite Coated with                                                                       >90%     >90%                                              Osteogenic Preparation                                                        Polylactic Acid Coated with                                                                      >90%     >90%                                              Osteogenic Preparation                                                        Hydroxyapatite Coated with                                                                       >75%     >90%                                              Osteogenic Preparation                                                        and Treated with                                                              Gamma-Irradiation                                                             ______________________________________                                    

EXAMPLE 8 Polyclonal Antisera Against Osteogenically Active Proteins p3OF 31-34

Antisera specific for proteins containing subunits A or D were generatedagainst the synthetic peptides obtained from Peninsula Laboratories,Belmont, Calif. The synthetic peptides comprised branched lysineheptamers to which were linked either eight peptides having the aminoacid sequence set out in SEQ ID NO: 29 or eight peptides having theamino acid sequence set out in SEQ ID NO: 30. The peptides were linkedto the heptamers by their carboxy termini.

    ______________________________________                                                                    Antibody                                                                      Designa-                                          Antigen                     tion                                              ______________________________________                                        SEQ ID NO: 29                                                                            SAPGRRRQQARNRSTPAQDV AbANt                                         Subunit A                                                                     SEQ ID NO: 30                                                                            STGGKRRSQNRSKTPKNQEA AbDNt                                         Subunit D                                                                     ______________________________________                                    

Antisera were generated in rabbits (3- to 6-month-old New Zealand whitemale) using standard procedures of subcutaneous injections, first incomplete Freunds adjuvant, and later (at 14 and 21 days) in incompleteFreunds adjuvant followed by bleeding and preparation of antisera.

The AbANt and AbDNt antisera were cross-reactive with the syntheticpeptide antigens when used in an ELISA format as described in Example14, and the reduced subunits A and D when used in a Western Blot formatas described in Example 13. The AbANt and AbDNt antisera were alsocross-reactive with the osteogenically active proteins P3 OF 31-34 whenused in either an ELISA or Western Blot format. These antisera were notcross-reactive with any presently defined form of subunit B or subunit Cas determined by Western Blot analysis against purified subunit B andsubunit C.

A fusion protein of E. coli ribolukinase fused to the 129 amino acidsequence of the carboxy-terminal region of BMP-2A (Wozney, et al.,Science, 242, 1528-1534 (1988)) was constructed, expressed and purifiedessentially as described by Lai, et al., PCT/US86/00131. A syntheticgene encoding the polypeptide designated BMP-2A and the 15 amino acidresidues preceding this sequence (Wozney, et al., Science, 242,1528-1534 (1988)) was constructed using oligonucleotides designed withcodons preferred by E. coli. This synthetic gene was cloned into aderivative of the pING1 vector, thereby yielding a fusion protein ofribulokinase fused to the 129 amino acid sequence of the carboxyterminal region of BMP-2A (homologous to subunit B). This construct wastransformed into E. coli strain MC1061 and, following induction with0.5% arabinose, yielded the ribulokinase-B fusion protein produced ininclusion bodies. Inclusion bodies were isolated and extensively washed,yielding a purified preparation of the ribulokinase-B fusion protein.

Antisera specific for subunit B were generated by formic acid cleavageof the ribulokinase-B fusion protein to produce separate ribulokinaseand subunit B proteins. The fusion protein was cleaved using 70% formicacid at 37° C. for 48 to 72 hours. The acid-cleaved proteins werelyophilized, reduced, and carboxymethylated. The subunit B was isolatedby passage through a Q-Sepharose column equilibrated at 50 mM MES pH6.5, 6M urea, conductivity 10 mS/cm; and then binding to an S-Sepharosecolumn using the same MES buffer. The subunit B bound to the S-Sepharoseand was eluted by 1.0M NaCl, desalted, and further purified onreverse-phase HPLC eluting in the range 35 to 45% acetonitrile. Theisolated subunit B was injected into rabbits as described above. Thisantisera is designated AbB. The AbB antisera was cross reactive with theisolated B subunit and with the osteogenically active proteins when usedin a Western format, as described in Example 13. This antisera was notcross reactive with any presently defined form of subunit A or D asdetermined by Western Blot.

EXAMPLE 9 Cloning of cDNA For Human Subunit D

A variety of techniques can be used to identify sequences of human DNAencoding proteins homologous to a particular sequenced protein. Suchmethods include the screening of human DNA, human genomic libraries andhuman cDNA libraries. A variety of oligonucleotide probes can be usedincluding probes exactly complementary to the human DNA sequence,mixtures of probes complementary to all or some of the possible DNAsequences coding for the particular protein sequence, degenerate probessynthesized such that all possible sequences complementary to allpossible DNA sequences coding for the particular protein sequence arerepresented, and degenerate probes synthesized using nucleotideanalogues such as deoxyinosine triphosphate. In this example, thepolymerase chain reaction (PCR) technique was used to amplify sequencesof human cDNA encoding proteins homologous to subunit D of bovineosteogenically active preparations P3 OF 31-34.

Preparation of cDNA From U-2 OS Cells

The human osteogenic sarcoma cell line U-2 OS was obtained from the ATCC(American Type Culture Collection, Rockville, MD) and maintained inMcCoy's 5a medium supplemented with 10% fetal calf serum and 1%glutamine/penicillin/streptomycin. Unless otherwise described, DNAmanipulations, definition of terms, and compositions of buffers andsolutions are described by Maniatis, T., et al., Molecular Cloning: ALaboratory Manual (1982). Poly (A)⁺ RNA was isolated from U-2 OS cellsusing the Fast Track-mRNA isolation kit from Invitrogen (San Diego,Calif.). A first strand cDNA copy of the mRNA was generated with oligo(dT) as the primer using the AMV Reverse Transcriptase System I fromBethesda Research Laboratories (BRL, Gaithersburg, Md.). Each reactionused 1 μg of poly (A)⁺ RNA which was reverse transcribed into firststrand cDNA that was used as template in eight separate polymerase chainreaction (PCR) DNA amplification reactions. Following cDNA synthesis,RNA was hydrolyzed by treatment with 50 mM NaOH at 65° C., followed byneutralization in 0.2N HCl.

PCR Amplification

Polymerase chain reaction (PCR), as described in R. K. Saiki, et al.,Science 239:487-491 (1988), was used to amplify DNA from U-2 OS cDNAprepared as described above. Oligonucleotide primers for PCR weresynthesized on an automated DNA synthesizer and were derived from theamino terminal and internal amino acid sequences of bovine subunit D.The 5' PCR primer, designated ODM-1, corresponded to sequence set out inSEQ ID NO: 31 of the first 11 amino acids from the amino terminus ofbovine subunit D, namely STGGKQRSQNR. This 32-mer contained all possiblecombinations of nucleotide sequence coding for this sequence of aminoacids and was greater than 4-million-fold degenerate. The nucleotidesequence of ODM-1 was

SEQ ID NO: 32

5'-[T/A][C/G]NACNGGNGGNAA[G/A]CA[G/A][C/A]GN[T/A][C/G]NCA[G/A]AA[C/T][C/A]G-3'.

Bracketed nucleotides are alternatives, and "N" means all alternatives(A, C, T and G).

The 3' PCR primer corresponded to an internal sequence of bovine subunitD set out in SEQ ID NO: 33, NHAIVQTLVHFIN, and was synthesized as theinverse and complementary sequence. This oligonucleotide primer wasdesignated ODB-1 and had the sequence

SEQ ID NO: 34

5'-TTTTTTTTGGATCC[G/A]TTXAT[G/A]AA[G/A]TGXACXA[G/A]XGT[C/T]TGXACXATXGC[G/A]TG[G/A]TT-3'.

Bracketed nucleotides are alternatives, and "X" represents thenucleotide analog deoxyinosinetriphosphate (dITP), which was used in allpositions where all four of the nucleotides (A, C, T or G) werepossible. (In the Sequence Listing for SEQ ID NO: 34 incorporatedherewith, dITP is designated as "N.") The sequence is preceded on the 5'end by a string of eight T's, followed by the sequence GGATCC whichdesignates a BamHI recognition site, leaving a stretch of 39 nucleotidescorresponding to the internal amino acid sequence of bovine subunit D.

Amplification of DNA sequences coding proteins homologous to bovinesubunit D using these two primers was accomplished using thePerkin-Elmer Cetus Gene Amp DNA Amplification Reagent Kit (obtainedeither from Parkin-Elmer Cetus, Norwalk, Conn., or United StatesBiochemical Corporation, Cleveland, Ohio). The PCR reaction contained 1μg of each primer ODM-1 and ODB-1, 1/8 of the synthesized U-2 OS firststrand cDNA (approximately 25-50 ng), 200 micro M of each dNTP, and 2.5UAmpli-Taq DNA Polymerase in the kit-supplied reaction buffer of 50 mMKCl, 1.5 mM MgCl₂, 0.1% (w/v) gelatin. PCR was performed for 30 cyclesconsisting of 1.5 minutes denaturation at 94° C., 2 minutes annealing at50° C. and 3 minutes elongation at 72° C. After the 30 cycles, a final10-minute elongation at 72° C. is performed.

The PCR products were analyzed by agarose gel electrophoresis, whichrevealed a major band of amplified DNA of approximately 300 bp. ASouthern Blot was performed in which the DNA in the gel was transferredto a Nytran nylon membrane (Schleicher and Schuell, Keene, N.H.) usingan LKB Vacugene Vacuum Blotting Unit, and then the DNA wasUV-crosslinked to the membrane using a Stratalinker (Stratagene,LaJolla, Calif.). The membrane was probed for amplified sequencesencoding proteins homologous to bovine subunit D using a probecorresponding to the amino acid sequence set out in SEQ ID NO: 35,KTPKNQEALR. This sequence is found near the amino terminus of bovinesubunit D, following the sequence used to construct the 5' PCR primer.This probe would therefore hybridize to amplified sequences that encodeproteins homologous to bovine subunit D without overlapping either ofthe two primers used in the amplification. This 29-mer probe wasdesignated ODibb and had the sequence

SEQ ID NO: 36

AAXACXCCXAA[G/A]AA[C/T]CAXGA[G/A]GC X[C/T]TX[C/A]G

where bracketed nucleotides are alternative and "X" represents dITP,which was used in positions where all four nucleotides (A, C, T or G)were possible. (In the Sequence Listing for SEQ ID NO: 36 incorporatedherewith, dITP is designated as "N.") The Southern Blot wasprehybridized at 42° C. in 5×SSPE (SSPE=0.18M NaCl, 0.01M NaH₂ PO₄,0.001M EDTA, pH 7.4), 0.5% SDS, 3× Denhardt's, 100 μg/ml salmon spermDNA, then hybridized at 42° C. in 6×SSPE, 0.5% SDS to the ODibb probewhich had been radioactively labelled using polynucleotide kinase andγ[³² P]ATP. The blot was washed at 42° C. in 2×SSC (SSC=0.15M NaCl,0.015M Na Citrate, pH 7.0), 0.1% SDS. Autoradiography of the blot showedthat ODibb hybridized specifically to the 300 bp PCR-amplified DNA.

5' phosphates were added to the blunt-ended PCR product using kinase andATP, and the DNA was then ligated into the SmaI cut (blunt end) site ofthe vector pT7T3 18U (Pharmacia, Piscataway, N.J.). Following digestionwith SmaI to linearize any relegated vector, the recombinant plasmid DNAwas used to transform E. coli TG1 cells. Several transformants werepicked and used to purify plasmid DNA by a minilysate procedure. Thesize of the insert contained in these plasmids was confirmed to be 300bp by restriction analysis.

Cloned cDNAs from seven different transformants were sequenced bydideoxy sequencing methods (Sequenase, United States Biochemical Corp.).The sequences of three of these clones were identical to each other and;when translated to amino acid sequence, it was confirmed that they werehomologous to the sequence of bovine subunit D.

Cloning and Nucleotide Sequence of cDNA for Human Mature D

cDNA libraries were constructed from poly (A)⁺ RNA isolated from thehuman osteosarcoma cell line U-2 OS in λgt10 vectors. Libraries wereconstructed using oligo (dT) as primer, using kits obtained either fromAmersham (cDNA) Synthesis System Plus and cDNA Cloning System λgt10) orInvitrogen (The Librarian X) according to manufacturers protocols. Atotal of approximately 850,000 recombinant plaques generated in twolibraries were screened with a [³² P]dCTP-labeled random-primergenerated probe designated OD. This OD probe was an approximately 300 bpfragment of DNA, amplified by PCR from one of the hOD clones, using PCRprimers corresponding to the exact sequences from the regions of theoriginal degenerate PCR primers, ODM-1 and ODB-1, which were present inthis clone. This OD probe therefore contained at least 214 bp of exactsequence for human subunit D. Duplicate nylon replica filters (Hybond N,Amersham) were hybridized with OD at 60° C. in 6×SSPE, 5× Denhardt's,0.5% SDS, 0.05 mg/ml sheared salmon sperm DNA for 16 to 40 hours,following a 1 hour prehybridization in the same solution without probe.Filters were washed two to three times for 10 minutes each at roomtemperature in 2×SSC, 0.1% SDS, followed by successive 1 hour washes at65° C. in 2×SSC, 0.1% SDS and 1×SSC, 0.1% SDS. Filters were subjected toautoradiography for 1 to 4 days. The OD probe hybridized to severalpositives which appeared on duplicate filters, three of which wereidentified by PCR (following plaque purification) to contain sequencescorresponding to human subunit D. The DNA inserts contained in thesethree phage clones were amplified by PCR, essentially as describedabove, using primers corresponding to the sequence of the λgt10 vectorflanking the inserts; namely, λgt10F with the sequence

SEQ ID NO: 37

5'-GAGCAAGTTCAGCCTGGTTAAGTCC-3'

and λgt10R with the sequence

SEQ ID NO: 38

5═-TGGCTTATGAGTATTTCTTCCAGGG-3'.

Southern Blots of PCR-amplified DNA were probed with an oligonucleotideprobe designated ODUC-1, labeled with [³² P]ATP and polynucleotidekinase, which corresponds to the reverse and complementary sequencebetween nucleotides 38 and 67, is specific for the sequence of subunitD, and has the sequence

SEQ ID NO: 39

5'-GTCGCTGCTGCTGTTCTCTGCCACGTTGGC-3'.

The PCR amplified DNA from the longest of these three cDNA clones wassubcloned in the plasmid vector pT7T3 18U and sequenced. This clonecontained the sequence of the entire coding region corresponding tohuman mature D. This sequence is shown in FIG. 6 along with thecorresponding amino acid sequence.

Cloning of human prepro D

A human placental cDNA library in λgtII (Clontech HL1075b) was screenedwith the random primer labelled OD probe as described above. Onepositive hybridizing plaque was identified that was 1.6kb long and thisclone was designated pOD601. This clone contained the entire codingregion of mature D and most of the coding region of prepro D, but wasstill short of full length by approximately 240 bp at the 5' end.

The remaining sequence encoding the 5' end of prepro D was obtained byPCR amplification essentially as described above, using the primersODP-Sal:

SEQ ID NO: 40

5'-GAATTCGTCGACATGCACGTGCGCTCA-3'

and ODPP-3:

SEQ ID NO: 41

5'-CCATGGCGTTGTACAGGTCCAG-3'.

OPD-Sal introduced a SalI site at the 5' end of prepro D, while ODPP-3corresponded to the sequence of prepro D near the 5' end of pOD601,encompassing a naturally occurring NcoI site.

EXAMPLE 10 Cloning of cDNA for Human Subunit B Cloning of Human Mature B

DNA comprising the sequence of 342 nucleotides representing human matureB was obtained by PCR amplification of DNA generated by reversetranscription of U-2 OS poly (A)+ RNA. The amino terminus of mature Bwas inferred from alignment of the human BMP-2A sequence with the aminoacid sequences of bovine A, B, C, and D, and the presence of a blockedamino terminal on bovine subunit B as described in Example 4, presumablyresulting from cyclization of an N-terminal glutamine to form anon-sequenceable pyroglutamic acid.

PCR primers corresponded to the sequences obtained from Wang, et al.,PCT US87/01537. The 5' PCR primer (a 27-mer designated OB-NM) encodedamino acids set out in SEQ ID NO: 42, QAKHKQRKR, and had the nucleotidesequence

SEQ ID NO: 43

5'-CAAGCCAAACACAAACAGCGGAAACGC-3'.

The 3' PCR primer (a 37-mer designated OB-CP) encoded amino acids setout in SEQ ID NO: 63, VEGCGCR, and was synthesized as the inverse andcomplementary sequence, preceded on the 5' end by a stop codon, an SstIIsite, and a HindIII site. The nucleotide sequence of OB-CP was

SEQ ID NO: 44

5'-AAGCTTCCGCGGCTAGCGACACCCACAACCCTCCACA-3'.

The sequence of PCR-amplified human mature B is shown in FIG. 7 and SEQID NOS: 3 and 4 along with the corresponding amino acid sequence.

Cloning of Human Prepro B

Cloning of cDNA encoding prepro B was accomplished by PCR amplificationfrom U2-OS mRNA essentially as described above, except that Vent DNApolymerase (New England Biolabs) was used instead of Taq polymerase, andthe denaturation step was carried out at 96° C. The primers used for PCRwere OB-PPN:

SEQ ID NO: 45

5'-ACTGTCGACATGGTGGCCGGGACCCG-3'

and OB-PPC:

SEQ ID NO: 46

5'-ACGTTTTTCTCTTTTGTGGAGAGGAT-3'

which were successfully used to amplify an 850 bp fragment correspondingto the entire coding region of prepo B.

EXAMPLE 11 Construction of Mammalian Expression Vectors for theProduction Of Human Subunits B and D

Plasmid vectors that contain cDNA genes for subunits B and D wereconstructed based on vectors originally developed for expression of theantibody heavy chain genes (Better, et al., PCT US89/03842): pING1714and pING2237N (which is a derivative of pING2227 containing a gene fordhfr selection and a unique NotI site). These vectors have a number offeatures useful for regulating gene expression in mammalian cells.

Transcriptional activity is controlled by the heavy chain mouse enhancerderived from M13 M8alphaRX12 (Robinson, et al., PCT US86/02269) locatedadjacent to the mouse Abelson virus LTR promoter/enhancer (Abl) derivedfrom pelin2 (provided by Dr. Owen Witte, UCLA, and described by Reddy,et al., Proc. Natl. Acad. Sci. USA), 80, 3623 (1983)) in pING2237N andby the Abl in pING1714. Downstream of the Abl promoter is the 16S splicedonor and acceptor segment from-SV40 in both vectors, which was excisedfrom pUC12/pL1 (Robinson, et al., PCT US86/02269). The expressed geneswere located just downstream of the splice junction. At the 3' end ofthe gene in pING2237N, the human genomic gamma-1 polyadenylationsequence has been cloned as a 1187-bp DNA fragment described by Ellison,et al., Nucl. Acids Res., 10, 4071 (1982).

The remainder of the vector is similar to pSV2, containing a selectablemarker (neo or dhfr) under the control of the SV40 early promoter andsequences of pBR322 necessary for growth in E. coli.Plasmid pING2237Ncontains a unique NotI site located in the pBR322-derived sequences thatwas introduced at the unique AatII site by cutting with AatII andligating the annealed oligonucleotides

SEQ ID NO: 47

5'-TGAAGCGGCCGCAACAGACGT-3'

and

SEQ ID NO: 48

5'-CTGTTGCGGCCGCTTCAACGT- 3'.

This created a vector that could be linearized with NotI prior totransfection into mammalian cells. The oligonucleotides were chosen, andresulting clones were selected such that the AatII site was regeneratedon one side of the NOtI site only.

A useful feature of the vector pING1714 (and pING2237N) is that itcontains unique SalI and SstII sites into which genes such as thoseencoding the subunits B and D can be cloned. The SalI site is positionedso that insertion at this site generates a transcriptional fusioncontrolled by the enhancer/promoter region. The SstII site is located inthe CH3 domain of the heavy chain gene.

Construction of Vectors for Expression of Subunits B and D in AnimalCells

DNA representing the entire protein region of subunit C (prepro plusmature) and consisting of 1224 nucleotides was obtained by PCRamplification of DNA generated by reverse transcription of U-2 OS poly(A)⁺ RNA. PCR primers corresponded to the sequences obtained from Wang,et al., PCT US87/01537. The 5' PCR primer (a 37-mer designated OC-NP)encoded amino acids set out in SEQ ID NO: 49, MIPGNRML, and was precededon the 5' end by a SalI site and an EcoRI site. OC-NP had the nucleotidesequence

SEQ ID NO: 50

5'-GAATTCGTCGACATGATTCCTGGTACCGAATGCTGA-3'.

The 3' PCR primer (a 37-mer designated OC-CP) encoded the amino acidsequence set out in SEQ ID NO: 51, VEGCGCR, and was synthesized as theinverse and complementary sequenced, preceded on the 5' end by a stopcodon, an SstII site, and a HindIII site. The nucleotide sequence ofOP-CP was

SEQ ID NO: 52

5'-AAGCTTCCGCGGCTCAGCGGCACCCACATCCCTCTACT-3'.

The sequence of PCR-amplified human prepro and mature C is shown in FIG.8 along with the corresponding amino acid sequence. The amino terminusof mature C is inferred from alignment of the human BMP-2B sequence withthe amino terminal sequence of bovine C. The PCR-amplified gene was cutwith SalI and SstII and cloned into pING1714. The resulting plasmidpING3900 still contained about 470 bp of DNA from the C-terminal domainof the heavy chain constant region between the end of the C gene and thegamma-1 polyadenylation sequence.

To eliminate these sequences and construct a vector with a unique NotIsite, pING3900 served as the template for the construction of two othervectors. A three-piece ligation was performed with the SalI to BglIIvector fragment (containing the NotI site) from pING2237N and the SalIto SstII and SstII to BglII fragments from pING3900, generatingpING3901. This vector then contained the C gene and a unique NotI site,yet still contained the heavy chain gene Segment. To remove thissegment, pING3901 was cut with AatII and BamHI and the vector fragmentwas isolated. Concurrently, pING3901 was cut with AatII and SstII, andthe fragment containing the enhancer/promoter, 16S splice and the C genewas purified. The genomic heavy chain polyadenylation region wasamplified from pING2237N by PCR with the primers

SEQ ID NO: 53

5'-ACTACCGCGGTAAATGAGTGCGACGG-3'

and

SEQ ID NO: 54

5'-CACTGCATTCTAGTTGTGGT-3'.

The former primer introduced an SstII site at its 5' end while thelatter primer is located in the vector sequences downstream of the BamHIsite. The PCR-amplified fragment was cut with SstII and BamHI, and thethree fragments were ligated to generate the plasmid pING3902.

Plasmid pING3902 contains unique SalI and SstII sites and was used toconstruct mammalian expression vectors for genes encoding subunits B andD. The gene sequences encoding the mature subunits B and D (FIG. 7 and6, respectively) were amplified by PCR to yield a blunt end at the 5'end and contain an SstII site just following the termination codon ofthe genes. Primers

SEQ ID NO: 55

5'-CAAGCCAAACACAAACAGCGGAAACGC-3'

and

SEQ ID NO: 56

5'AAGCTTCCGCGGCTAGCGACACCCACAACCCTCCACA-3'

were used to amplify the coding sequence for mature B. Primers

SEQ ID NO: 57

5'-TCCACGGGGAGCAAACAGCGCA-3'

and

SEQ ID NO: 58

5'-CATACCGCGGAGCTAGTGGCAGCCACA-3'

were used to amplify the coding sequence for mature D. These fragmentswere each digested with SstII.

Likewise, the prepro C gene segment (ppC) shown in FIG. 8 was amplifiedfrom first-strand cDNA from U-2 OS mRNA by PCR with primers

SEQ ID NO: 59

5'-GAATTCGTCGACATGATTCCTGGTAACCGAATGCTGA-3'

and

SEQ ID NO: 60

5'-ACGCTTGGCCCTCCGGCGTCGGGTCAA-3'

so that it contained a SalI restriction site just upstream of theinitiation codon ATG and a blunt end at its 3' end. This fragment wasdigested with SalI.

A three piece ligation with the prepro C gene fragment, the mature Bgene fragment and the purified vector fragment of pING3902, resultingfrom digestion with SalI and SstII, yielded plasmid pING3904 containingthe ppC-mature B gene.

A three piece ligation with the prepro C gene fragment, the mature Dgene fragment and the purified vector fragment of pING390.2, resultingfrom digestion with SalI and SstII, yielded plasmid pING3906 containingthe ppC-mature D gene.

Construction of Vectors with an Alternate Drug Resistance Gene.

Additional vectors were constructed for mammalian gene expression thatdiffer from those described above, only in the selectable drugresistance gene. Plasmid pING3005 is similar to pING1714 except that itcontains the xanthine-guanine phosphoribosyl transferase (gpt) geneinstead of the neomycin phosphotransferase gene (neo). Plasmid pING3906was cut with BglII, treated with calf intestinal alkaline phosphatase(CIAP), cut with SalI, and the vector fragment was purified. PlasmidpING3005 was cut with BamHI plus BglII, and the DNA fragment containingthe gpt gene was purified.

These two fragments were ligated to the purified B and D gene fragmentsfrom pING3904 and pING3906, respectively, that had been excised withBamHI, treated with CIAP, and cut with SalI. These ligations generatedplasmids pING3918 (B) and pING3919 (D) both containing the gptselectable marker.

These plasmids were transfected into mammalian cells along with vectorscontaining the neo marker either together or sequentially to generatecell lines producing combinations of heterologous genes.

Construction of Vectors for the Expression of B and D With HomologousPrepro Sequences

To construct prepro B-mature B expression vectors, the 850 bp fragmentcorresponding to prepro B described above was digested With SalI, a siteintroduced by the 5'PCR primer, and NcoI, a site within the preprosequence; the resulting 680 bp fragment was purified. To obtain afragment containing the remainder of the prepro and the mature sequenceof B, a DNA fragment was PCR amplified from U2-OS mRNA using the primersPPOB-2, located upstream of the NcoI site

SEQ ID NO: 61

(primer sequence 5'-TTTTTTCCAGT CTTTTGGACACCAGGTTGG-3'),

and OB-CP, which introduced an SstII site at the end of the matureregion

SEQ ID NO: 62

(primer sequence 5'-AAGCTTCCGCGGCTAG CGACACCCACAACCCTCCACA-3').

This fragment was digested with NcoI and SstII and purified. A 3-pieceligation was performed with the SalI-NcoI and NcoI-SstII fragments justdescribed and the SalI-SstII vector fragment from pING3920 (identical tothe corresponding vector segment from pING3902) to generate pING4207(gpt gene). To construct a vector with dhfr instead of gpt, theClaI-DraIII fragment of pING4207 containing the entire B gene was clonedinto the ClaI-DraIII vector fragment of pMB27, a vector essentiallysimilar to pING3902 except that it contained the dhfr gene. Theresulting construct was called pING4206.

To construct prepro D- mature D expression vectors, the following 4fragments were ligated together: (1) the 270 bp SalI-NcoI fragment fromthe 5'end of prepro D, generated by PCR using primers ODP-Sal and ODPP-3as described above; (2) an 850 bp NcoI-AlwNI fragment from the cDNAclone pOD601; (3) a 150 bp AlwNI-SstII fragment corresponding to the endof mature D excised from pING3919; and (4) the SalI-SStII vectorfragment from pING3920. The resulting vector was designated pING4120 andhad gpt marker.

To construct a single vector for the simultaneous coexpression of B andD, plasmid pING4206 (B gene) was linearized at the unique AatII site andligated to an AatII fragment from pING4120 containing the entire D gene,yielding the 2-gene vector pING4121 (dhfr) .

EXAMPLE 12 Expression of Human Osteogenically Active Proteins fromAnimal Cells

According to this example, various osteogenically active proteins wereexpressed from animal cells including B and D monomers, B/B and D/Dhomodimers and the B/D heterodimer.

Stable Transfection of CHO K-1 Cells for the Production Of Homodiners ofHuman Subunits D or B

The cell line CHO K-1 (ATCC CRL 61) was grown in Ham's F12 medium plus10% fetal bovine serum. The medium is supplemented withglutamine/penicillin/streptomycin (Irvine Scientific, Irvine, Calif.).

The cells were transfected using the calcium phosphate method of Wigler,et al., Cell, 11, 223 (1977). Following the calcium phosphate treatment,the cells are plated in T150 flasks, and transfectants were obtained bygrowth in the presence of selective medium. Untransformed cells wereremoved during successive feedings with selective medium and, at 10 daysto 2 weeks, only microcolonies of transfected cells were observed. G418selection was used at 0.6 mg/ml. Mycophenolic acid was used at 24 μg/mlplus 0.25 mg/ml xanthine.

Cell lines producing subunit D or B were obtained as described below.The expression plasmids pING3906 or pING3904 was digested with NOtI andtransfected separately into the CHO K-1 cells to yield G418-resistantcells. The transfectants were grown in T-flasks, trypsinized andsubcloned. The subclones were screened for the presence of D-specific orB-specific messenger RNA isolated as described by White, J. Biol. Chem.,57, 8569 (1982) or Gough, Anal. Biochem., 173, 93 (1988), and probed onslot blots with ³² [P]-labeled D-specific or B-specific DNA.

Those cell lines that were identified as producing the highest levels ofmRNA were expanded and grown in Ham's F12 medium containing 10% FBS andthen shifted into serum-free (HB-CHO, Irvine Scientific) or protein-freemedium (PFHM, Gibco) for the production of D or B.

Stable Transfection of CHO K-1 Cells for the Production of Mixtures ofHuman Subunits D and B

According to the invention, cell lines to be transformed with genesencoding both subunit B and subunit D can be transformed according to avariety of methods including, but not necessarily limited to (1)co-transformation with two genes on two vectors at once (2) sequentialtransformation first with one gene and then another; and (3)transformation with two genes on the same vector.

To obtain cell lines which produce mixtures of subunits D and B, clonesof D-producing transfectants which were transfected with the plasmidcontaining the neo selectable marker (pING3906) were transfectedaccording to the calcium phosphate method with the plasmid constructedwith the gpt selective marker containing the gene encoding the B subunit(pING3918). G418- and MPA-resistant transfectants were then screened forthe production of B- and D-specific mRNA. Cell lines that expressed bothmRNAs were grown in large volumes in Ham's F12 plus 10% FBS and thenshifted into serum-free or protein-free medium for the purpose ofproducing B/D dimers. One such cell line has been designated C1131.

An alternative method to obtain cell lines which produce mixtures ofsubunits B and D, involved co-transfection of NotI-digested pING3904 andpING3906 into CHO cells to give G418-resistant cells. Transfectants weregrown in T-flasks, trypsinized and subcloned. The subclones werescreened for the presence of B- and D-specific messenger RNA. Those celllines that produce both messages were scaled up for the production ofB/D dimers.

Stable Transfection of Mouse Lymphoid Cells For the Production ofHomodimers of Human Subunits B or D, and a Mixture of Subunits B and D

The cell line Sp2/0 (American Type Culture Collection CRL 1581]was grownin Dulbecco's Modified Eagle Medium plus 4.5 g/l glucose (DMEM, Gibco)plus 10% fetal bovine serum. The medium was supplemented withglutamine/penicillin/streptomycin (Irvine Scientific, Irvine, Calif.).

The electroporation method of Potter, et al., Proc. Nat. Acad. Sci.(USA), 81, 7161 (1984) was used. After transfection, cells were allowedto recover in complete DMEM for 24 to 48 hours, and then seeded eitherinto 96-well culture plates at 10,000 to 50,000 cells per well or inT-flasks at 5×10⁴ cells/ml in the presence of selective medium. G418(Gibco) selection was used at 0.8 to 1.2 mg/ml. Mycophenolic acid (MPA,Calibiochem) was used at 6 mg/ml plus 0.25 mg/ml xanthine. Theelectroporation technique gave a transfection frequence of 1 to 10×10⁻⁵for the Sp2/0 cells.

Cell lines producing subunit D were obtained as described below. Theexpression plasmid pING3906 (containing the neo selectable marker) wasdigested with NotI and transfected into the Sp2/0 cells. Approximately75% of the cells were plated into 96-well plates. The remaining 25% wereplated into T25 or T75 flasks. Clones of D-producing transfectants werescreened directly for the presence of D-specific messenger RNA.

Those cell lines that were identified as producing the highest levels ofmRNA were expanded and grown either in DMEM medium plus 10% fetal bovineserum or in protein-free medium (PFHM, Gibco) for the production ofsubunit D. By this strategy, cell lines which produce subunit B orhomodimers of subunit B were similarly developed.

To obtain cell lines which produce mixtures consisting of subunits D andB, a D-producing cell line which had been transfected with a plasmidcontaining the neo selectable marker (pING3906) was subsequentlytransfected with a plasmid containing the gpt selective marker and thegene encoding the B subunit (pING3918). G418- and MPA-resistanttransfectants were then screened for the production of B- and D-specificmRNA. Cell lines that express both mRNAs were grown in serum-free orprotein-free medium.

EXAMPLE 13 Detection of Subunits B and D Using Western Blot Assay

Protein samples were electrophoresed on 12.5 or 15% SDS polyacrylamidegels (SDS-PAGE), and electrophoretically transferred to eitherpolyvinylidine difluoride (PVDF) transfer membrane or nitrocellulose inthe presence of 10% methanol, 10 mM cyclohexylamino-1-propanesulfonicacid (CAPS), pH 10-11, at 0.5 amp for 15 to 30 minutes. The PVDFmembrane or nitrocellulose filter was treated for Western Blot analysisutilizing antibodies generated as described in Example 8.

The PVDF membrane or nitrocellulose paper containing the protein wasplaced in a solution-designated buffer P (composed of 20 mM phosphate,pH 7.4; 0.15M NaCl; 0.05% Tween-20; 0.25% gelatin; and 0.02% sodiumazide) for a minimum of 1 hour at 22° C. with agitation.

Buffer P was then replaced by buffer Q (composed of buffer P plusantibodies) for a minimum of 1 hour at 22° C. (or overnight at 4° C.).Buffer Q was replaced by buffer P, which was changed four times over aminimum of 1 hour. Buffer P was replaced by buffer R (buffer P plus ¹²⁵I protein A at 2.5×10⁵ cpm/ml, Amersham) and incubated for 1 hour at-22° C. with agitation. Buffer R was replaced by buffer P, which waschanged at least four times during 1 hour of incubation.

The moist PVDF membrane or nitrocellulose filter was placed betweensheets of plastic wrap, and together with a lighting screen and X-rayfilm (Dupont Cronex, Wilmington, Del.), enclosed in a light-prooffolder, and placed at -70° C. for an appropriate period of time. Theexposed film was developed using standard techniques and equipment.

EXAMPLE 14 Detection of Subunit B and Subunit D Using Enzyme-LinkedImmunosorbant Assay-ELISA

ELISA assays were performed in 96-well Immulon plates (Dynatech) intowhich samples at several dilutions and in a final volume of 200 μlcontaining 3M urea, 15 mM Na₂ CO₃, 24 mM NaHCO pH 9.6 were bound.Binding was performed first at 60° C. for 15 minutes and subsequently at21° C. to 24° C. for 2 hours or at 4° C. for 12 to 18 hours in ahumidified chamber. Following binding, the wells of the plate wereindividually washed three times with a solution containing 8 mM Na₂HPO₄, 1.5 mM KH₂ PO₄, 2.7 mM KCl, 137 mM NaCl, and 0.05% Tween-20 inMillipore-filtered, distilled water (solution E), and then washed twotimes with Millipore-filtered, distilled water.

Antibody against the N-terminal of subunit D (described in Example 8),or against reduced and carboxymethylated subunit B (described in Example8) were added at a 1:1000 to 1:5000 dilution in solution E and incubatedat 21° C. to 24° C. for 2 hours. Following incubation with antibody, theplate was washed as described above and peroxidase-conjugated Goatanti-Rabbit antibody (Cappel) at 1:1000 dilution in solution E was addedto the plate and incubated at 21° C. to 24° C. for 2 hours. The platewas washed as described above and developed using the TMB reagent(Pierce) according to the manufacturer's instructions.

Typical assays contained recombinant protein prepared as described inExample 15, a positive control containing aliquots from a single lot ofa Prep-HPLC pool of bovine bone (described in Example 1), andappropriate negative controls. For purposes of comparison between thevarious recombinant protein preparations, the B-immunoreactivity and theD-immunoreactivity contained in 50 μg of the Prep HPLC pool of thebovine bone preparation was defined as 2 units of reactivity.

EXAMPLE 15 Characterization of Human Osteogenically Active ProteinsExpressed in Animal Cells

The osteogenically active proteins contained in the culture supernatantwere enriched using column chromatography steps as described inExample 1. For example, the conditioned media was adjusted to 6 M urea,50 mM MES, pH 6.5, conductivity 10 mS/cm (by addition of crystallineurea and 1M MES, pH 6.5). The adjusted sample was applied onto aQ-Sepharose column equilibrated in 6M urea, 50 mM MES, pH 6.5,conductivity 10 mS/cm. The unbound protein of the Q-Sepharose column wasapplied to a S-Sepharose column equilibrated in 6M urea, 50 mM MES, pH6.5, conductivity 10 mS/cm, and the S-Sepharose column was washed withthe same buffer to remove unbound protein. The protein bound to theS-Sepharose column was eluted with 6M urea, 50 mM MES, pH 6.5, 1M NaCl.Further enrichment was achieved using hydrophobic interactionchromatography on a Phenyl-Sepharose column. The sample in 6M urea, 50mM MES, 1M NaCl was made 50 mM in Tris HCl, pH 7.3-8.0, using 1M Trisstock, and was 25% saturated in ammonium sulfate using 13.4 g ammoniumsulfate for each 100 ml of sample. The sample was applied to aPhenyl-Sepharose column equilibrated in 6 M urea, 50 mM Tris HCl, pH7.3-8.0, 25% saturated ammonium sulfate, and the column was washed withthe same buffer to remove unbound protein. The bound protein was elutedusing 6M urea, 50 mM Tris HCl, pH 7.3 to 8.0 or 6M urea, 50 mM MES pH6.5. The sample was desalted and further purified by reverse phasechromatography using C-18 HPLC columns (as shown in Example 1)equilibrated with a buffer containing, by volume, 70% Buffer A and 30%Buffer B, as described previously (Buffer A is 0.05% trifluoroaceticacid in water and Buffer B is 0.025% trifluoroacetic acid inacetonitrile). Bound proteins were eluted using a linear gradient of 30%to 60% acetonitrile. The immunoreactive dimers eluted within theconcentrations of 35% to 45% acetonitrile, and were concentrated bylyophilization. Enriched samples isolated from the supernatant of cellstransfected with both the gene sequence for human mature B and the genesequence for human mature D (C1131) are called Prep B/D. Enrichedsamples isolated from the supernatant of cells transfected only with thegene sequence for human mature B are called Prep B. Enriched samplesisolated from the supernatant of cells transfected only with the genesequence for human mature D are called Prep D. Enriched samplescontaining admixtures of Prep B and Prep D are called Prep B+D.

Following lyophilization, the enriched samples were solubilized inMillipore-filtered, distilled water and were characterized for thepresence of subunit B and/or subunit D using specific antisera(described in Example 8), Western blot techniques (described in Example13), and ELISA assays (described in Example 14).

Western Blot analyses of the enriched samples isolated from media of CHOcells transfected with both the B-subunit sequence and the D-subunitsequence (Prep B/D), or separately transfected with only the D-subunitsequence (Prep D), or with only the B-subunit sequence (Prep B) areshown in FIG. 9. The Western blot analysis of the reduced samplesperformed with antibody specific for subunit B showed, in Prep B/D, abroad B-specific band of 16,000 to 17,000 daltons. Similar analysis ofPrep B showed an identical B-specific band. Analysis of reduced samplesusing D-specific antibody showed a broad D-specific band at 17,000 to19,000 daltons and a less prominent band at 22,000 to 23,000 daltons forsamples of Prep B/D and Prep D. Analysis of non-reduced Prep B/D andnon-reduced Prep D with D-specific antibody showed prominentD-containing dimers of 30,000 to 32,000 daltons and less prominentdimers extending to a molecular weight of approximately 37,000 daltons.Poor reactivity of the B-specific antibody with non-reduced Prep B andnon-reduced Prep B/D hindered Western blot demonstration of theB-subunit in Prep B and in Prep B/D (data not shown).

Polyacrylamide gel electrophoresis of the proteins contained inindividual HPLC fractions of Prep B/D, transfer of the proteins to PVDFmembrane (described in Example 13), and Coomassie staining were used toisolate the non-reduced, 30,000 to 32,000 dalton band. The excised bandwas sequenced using the gas phase sequenator. The sequence obtainedrevealed the N-terminal sequence STGSKQR-QN of the D-subunit and theN-terminal sequence QAK-KQR-L of the B-subunit (the complete sequencesof the D and B subunits are set out in SEQ ID NOS: 2 and 4,respectively).

The biological activity of the enriched samples of Prep B/D, Prep B+D,Prep D and Prep B were determined using the rat implant assay asdescribed in Example 2. ELISA assays were used to quantitate the amountof B-subunit or D-subunit immunoreactivity present in each of theenriched preparations. Comparison was made to the amount ofB-immunoreactivity and/or the amount of D-immunoreactivity contained in50 μg of the Prep-HPLC pool of the bovine bone preparation which isdefined as 2 units of immunoreactivity. Two units of the Prep HPLC poolof the bovine bone preparation contain 1 unit of B-immunoreactivity and1 unit of D-immunoreactivity, and, upon implantation for 21 days yielded14% of the area of the stained sections occupied by osteoid activity.Enriched samples of Prep B/D, Prep B+D, Prep B and Prep D containingvarious amounts of immunoreactivity were implanted for 17 days andanalyzed for percent bone formation in the explant tissue (FIG. 10). Themost potent bone formation activity from media of cells was obtainedwith Prep B/D such as from cell line Cl131. For example, implantation ofPrep B/D containing 1.5 units B-immunoreactivity and 0.8 units ofD-immunoreactivity, resulted in 55% of the area of the stained sectionsoccupied by osteoid activity. In contrast, implantation of Prep Bcontaining 1.5 units of B-immunoreactivity resulted in less that 10% ofthe area of the stained sections occupied by osteoid activity,implantation of Prep D containing 1.1 units of D-immunoreactivityresulted in only 1% of the area of the stained sections occupied byosteoid activity and implantation of an admixture of Prep B (1.5 unitsand Prep D (0.8 units) containing a total of 2.3 units resulted in only1% of the area of the stained sections occupied by osteoid activity.

Numerous modifications and variations in the practice of the inventionare expected to occur to those skilled in the art upon consideration ofthe foregoing descriptions of preferred embodiments thereof.Consequently, only such limitations should be placed upon the inventionas appear in the following claims.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 63                                                 (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 417 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..417                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       TCCACGGGGAGCAAACAGCGCAGCCAGAACCGCTCCAAGACGCCCAAG48                            SerThrGlySerLysGlnArgSerGlnAsnArgSerLysThrProLys                              15 1015                                                                       AACCAGGAAGCCCTGCGGATGGCCAACGTGGCAGAGAACAGCAGCAGC96                            AsnGlnGluAlaLeuArgMetAlaAsnValAlaGluAsnSerSerSer                              20 2530                                                                       GACCAGAGGCAGGCCTGTAAGAAGCACGAGCTGTATGTCAGCTTCCGA144                           AspGlnArgGlnAlaCysLysLysHisGluLeuTyrValSerPheArg                              35 4045                                                                       GACCTGGGCTGGCAGGACTGGATCATCGCGCCTGAAGGCTACGCCGCC192                           AspLeuGlyTrpGlnAspTrpIleIleAlaProGluGlyTyrAlaAla                              5055 60                                                                       TACTACTGTGAGGGGGAGTGTGCCTTCCCTCTGAACTCCTACATGAAC240                           TyrTyrCysGluGlyGluCysAlaPheProLeuAsnSerTyrMetAsn                              657075 80                                                                     GCCACCAACCACGCCATCGTGCAGACGCTGGTCCACTTCATCAACCCG288                           AlaThrAsnHisAlaIleValGlnThrLeuValHisPheIleAsnPro                              859 095                                                                       GAAACGGTGCCCAAGCCCTGCTGTGCGCCCACGCAGCTCAATGCCATC336                           GluThrValProLysProCysCysAlaProThrGlnLeuAsnAlaIle                              100105 110                                                                    TCCGTCCTCTACTTCGATGACAGCTCCAACGTCATCCTGAAGAAATAC384                           SerValLeuTyrPheAspAspSerSerAsnValIleLeuLysLysTyr                              115120 125                                                                    AGAAACATGGTGGTCCGGGCCTGTGGCTGCCAC417                                          ArgAsnMetValValArgAlaCysGlyCysHis                                             130135                                                                        (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 139 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       SerThrGlySerLysGlnArgSerGlnAsnArgSerLysThrProLys                              151015                                                                        A snGlnGluAlaLeuArgMetAlaAsnValAlaGluAsnSerSerSer                             202530                                                                        AspGlnArgGlnAlaCysLysLysHisGluLeuTyrValSerPheArg                              35 4045                                                                       AspLeuGlyTrpGlnAspTrpIleIleAlaProGluGlyTyrAlaAla                              505560                                                                        TyrTyrCysGluGlyGluCysAlaPheProLeu AsnSerTyrMetAsn                             65707580                                                                      AlaThrAsnHisAlaIleValGlnThrLeuValHisPheIleAsnPro                              8590 95                                                                       GluThrValProLysProCysCysAlaProThrGlnLeuAsnAlaIle                              100105110                                                                     SerValLeuTyrPheAspAspSerSerAsnValIleLeuLysLysTyr                              115120125                                                                     ArgAsnMetValValArgAlaCysGlyCysHis                                             130135                                                                        (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 342 base pairs                                                    (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                     (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..342                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       CAAGCCAAACACAAACAGCGGAAACGCCTTAAGTCCAGCTGTAAGAGA48                            GlnAlaLysHisLysGln ArgLysArgLeuLysSerSerCysLysArg                             151015                                                                        CACCCTTTGTACGTGGACTTCAGTGACGTGGGGTGGAATGACTGGATT96                            HisProLeuTyrValAs pPheSerAspValGlyTrpAsnAspTrpIle                             202530                                                                        GTGGCTCCCCCGGGGTATCACGCCTTTTACTGCCACGGAGAATGCCCT144                           ValAlaProProGlyTyrH isAlaPheTyrCysHisGlyGluCysPro                             354045                                                                        TTTCCTCTGGCTGATCATCTGAACTCCACTAATCATGCCATTGTTCAG192                           PheProLeuAlaAspHisLeuAsn SerThrAsnHisAlaIleValGln                             505560                                                                        ACGTTGGTCAACTCTGTTAACTCTAAGATTCCTAAGGCATGCTGTGTC240                           ThrLeuValAsnSerValAsnSerLysIlePro LysAlaCysCysVal                             65707580                                                                      CCGACAGAACTCAGTGCTATCTCGATGCTGTACCTTGACGAGAATGAA288                           ProThrGluLeuSerAlaIleSerMetLe uTyrLeuAspGluAsnGlu                             859095                                                                        AAGGTTGTATTAAAGAACTATCAGGACATGGTTGTGGAGGGTTGTGGG336                           LysValValLeuLysAsnTyrGlnAspM etValValGluGlyCysGly                             100105110                                                                     TGTCGC342                                                                     CysArg                                                                        (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 114 amino acids                                                  (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       GlnAlaLysHisLysGlnArgLysArgLeuLysSerSerCysLysArg                              1510 15                                                                       HisProLeuTyrValAspPheSerAspValGlyTrpAsnAspTrpIle                              202530                                                                        ValAlaProProGlyTyrHisAlaPheTyrCysHisGlyGluCysPro                               354045                                                                       PheProLeuAlaAspHisLeuAsnSerThrAsnHisAlaIleValGln                              505560                                                                        ThrLeuValAsnSerValAsnSerLysI leProLysAlaCysCysVal                             65707580                                                                      ProThrGluLeuSerAlaIleSerMetLeuTyrLeuAspGluAsnGlu                              8590 95                                                                       LysValValLeuLysAsnTyrGlnAspMetValValGluGlyCysGly                              100105110                                                                     CysArg                                                                        (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1224 base pairs                                                    (B) TYPE: nucleic acid                                                       (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..1224                                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       ATGATTCCTGGTAACCGAATGCTGATGGTCGTTTTATTATGCCAAGTC48                             MetIleProGlyAsnArgMetLeuMetValValLeuLeuCysGlnVal                             151015                                                                        CTGCTAGGAGGCGCGAGCCATGCTAGTTTGATACCTGAGACGGGGAAG96                             LeuLeuGlyGlyAlaSerHisAlaSerLeuIleProGluThrGlyLys                             202530                                                                        AAAAAAGTCGCCGAGATTCAGGGCCACGCGGGAGGACGCCGCTCAGGG144                           Ly sLysValAlaGluIleGlnGlyHisAlaGlyGlyArgArgSerGly                             354045                                                                        CAGAGCCATGAGCTCCTGCGGGACTTCGAGGCGACACTTCTGCAGATG192                           GlnSerH isGluLeuLeuArgAspPheGluAlaThrLeuLeuGlnMet                             505560                                                                        TTTGGGCTGCGCCGCCGCCCGCAGCCTAGCAAGAGTGCCGTCATTCCG240                           PheGlyLeuArgArg ArgProGlnProSerLysSerAlaValIlePro                             65707580                                                                      GACTACATGCGGGATCTTTACCGGCTTCAGTCTGGGGAGGAGGAGGAA288                           AspTyrMetArg AspLeuTyrArgLeuGlnSerGlyGluGluGluGlu                             859095                                                                        GAGCAGATCCACAGCACTGGTCTTGAGTATCCTGAGCGCCCGGCCAGC336                           GluGlnIleHi sSerThrGlyLeuGluTyrProGluArgProAlaSer                             100105110                                                                     CGGGCCAACACCGTGAGGAGCTTCCACCACGAAGAACATCTGGAGAAC384                           ArgAlaAsnThrV alArgSerPheHisHisGluGluHisLeuGluAsn                             115120125                                                                     ATCCCAGGGACCAGTGAAAACTCTGCTTTTCGTTTCCTCTTTAACCTC432                           IleProGlyThrSerGlu AsnSerAlaPheArgPheLeuPheAsnLeu                             130135140                                                                     AGCAGCATCCCTGAGAACGAGGCGATCTCCTCTGCAGAGCTTCGGCTC480                           SerSerIleProGluAsnGluAlaIle SerSerAlaGluLeuArgLeu                             145150155160                                                                  TTCCGGGAGCAGGTGGACCAGGGCCCTGATTGGGAAAGGGGCTTCCAC528                           PheArgGluGlnValAspGlnGl yProAspTrpGluArgGlyPheHis                             165170175                                                                     CGTATAAACATTTATGAGGTTATGAAGCCCCCAGCAGAAGTGGTGCCT576                           ArgIleAsnIleTyrGluValM etLysProProAlaGluValValPro                             180185190                                                                     GGGCACCTCATCACACGACTACTGGACACGAGACTGGTCCACCACAAT624                           GlyHisLeuIleThrArgLeuLeu AspThrArgLeuValHisHisAsn                             195200205                                                                     GTGACACGGTGGGAAACTTTTGATGTGAGCCCTGCGGTCCTTCGCTGG672                           ValThrArgTrpGluThrPheAspValSer ProAlaValLeuArgTrp                             210215220                                                                     ACCCGGGAGAAGCAGCCAAACTATGGGCTAGCCATTGAGGTGACTCAC720                           ThrArgGluLysGlnProAsnTyrGlyLeuAlaIleGl uValThrHis                             225230235240                                                                  CTCCATCAGACTCGGACCCACCAGGGCCAGCATGTCAGGATTAGCCGA768                           LeuHisGlnThrArgThrHisGlnGlyGlnHisV alArgIleSerArg                             245250255                                                                     TCGTTACCTCAAGGGAGTGGGAATTGGGCCCAGCTCCGGCCCCTCCTG816                           SerLeuProGlnGlySerGlyAsnTrpAlaGln LeuArgProLeuLeu                             260265270                                                                     GTCACCTTTGGCCATGATGGCCGGGGCCATGCCTTGACCCGACGCCGG864                           ValThrPheGlyHisAspGlyArgGlyHisAlaLeu ThrArgArgArg                             275280285                                                                     AGGGCCAAGCGTAGCCCTAAGCATCACTCACAGCGGGCCAGGAAGAAG912                           ArgAlaLysArgSerProLysHisHisSerGlnArgAlaAr gLysLys                             290295300                                                                     AATAAGAACTGCCGGCGCCACTCGCTCTATGTGGACTTCAGCGATGTG960                           AsnLysAsnCysArgArgHisSerLeuTyrValAspPheSerAspVal                               305310315320                                                                 GGCTGGAATGACTGGATTGTGGCCCCACCAGGCTACCAGGCCTTCTAC1008                          GlyTrpAsnAspTrpIleValAlaProProGlyTyrGlnAlaPhe Tyr                             325330335                                                                     TGCCATGGGGACTGCCCCTTTCCACTGGCTGACCACCTCAACTCAACC1056                          CysHisGlyAspCysProPheProLeuAlaAspHisLeuAsnSer Thr                             340345350                                                                     AACCATGCCATTGTGCAGACCCTGGTCAATTCTGTCAATTCCAGTATC1104                          AsnHisAlaIleValGlnThrLeuValAsnSerValAsnSerSerIl e                             355360365                                                                     CCCAAAGCCTGTTGTGTGCCCACTGAACTGAGTGCCATCTCCATGCTG1152                          ProLysAlaCysCysValProThrGluLeuSerAlaIleSerMetLeu                               370375380                                                                    TACCTGGATGAGTATGATAAGGTGGTACTGAAAAATTATCAGGAGATG1200                          TyrLeuAspGluTyrAspLysValValLeuLysAsnTyrGlnGluMet                              385 390395400                                                                 GTAGTAGAGGGATGTGGGTGCCGC1224                                                  ValValGluGlyCysGlyCysArg                                                      405                                                                           (2) INFORMATION FOR SEQ ID NO:6:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 408 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                       MetIleProGlyAsnArgMetLeuMetValValLeuLeuCysGlnVal                              1510 15                                                                       LeuLeuGlyGlyAlaSerHisAlaSerLeuIleProGluThrGlyLys                              202530                                                                        LysLysValAlaGluIleGlnGlyHisAlaGlyGlyArgArgSerG ly                             354045                                                                        GlnSerHisGluLeuLeuArgAspPheGluAlaThrLeuLeuGlnMet                              505560                                                                        PheGlyLeuArgArgArgPro GlnProSerLysSerAlaValIlePro                             65707580                                                                      AspTyrMetArgAspLeuTyrArgLeuGlnSerGlyGluGluGluGlu                              85 9095                                                                       GluGlnIleHisSerThrGlyLeuGluTyrProGluArgProAlaSer                              100105110                                                                     ArgAlaAsnThrValArgSerPheHisHisGluGl uHisLeuGluAsn                             115120125                                                                     IleProGlyThrSerGluAsnSerAlaPheArgPheLeuPheAsnLeu                              130135140                                                                     SerSerIle ProGluAsnGluAlaIleSerSerAlaGluLeuArgLeu                             145150155160                                                                  PheArgGluGlnValAspGlnGlyProAspTrpGluArgGlyPheHis                               165170175                                                                    ArgIleAsnIleTyrGluValMetLysProProAlaGluValValPro                              180185190                                                                     GlyHisLeuIleThrArgLeuLeu AspThrArgLeuValHisHisAsn                             195200205                                                                     ValThrArgTrpGluThrPheAspValSerProAlaValLeuArgTrp                              210215220                                                                     ThrArgGluLysGlnProAsnTyrGlyLeuAlaIleGluValThrHis                              225230235240                                                                  LeuHisGlnThrArgThrHisGlnGlyGlnHisValArgIleSerArg                               245250255                                                                    SerLeuProGlnGlySerGlyAsnTrpAlaGlnLeuArgProLeuLeu                              260265270                                                                     ValThrPheGly HisAspGlyArgGlyHisAlaLeuThrArgArgArg                             275280285                                                                     ArgAlaLysArgSerProLysHisHisSerGlnArgAlaArgLysLys                              290295 300                                                                    AsnLysAsnCysArgArgHisSerLeuTyrValAspPheSerAspVal                              305310315320                                                                  GlyTrpAsnAspTrpIleValAlaProProGlyTyrGln AlaPheTyr                             325330335                                                                     CysHisGlyAspCysProPheProLeuAlaAspHisLeuAsnSerThr                              340345350                                                                     A snHisAlaIleValGlnThrLeuValAsnSerValAsnSerSerIle                             355360365                                                                     ProLysAlaCysCysValProThrGluLeuSerAlaIleSerMetLeu                              370 375380                                                                    TyrLeuAspGluTyrAspLysValValLeuLysAsnTyrGlnGluMet                              385390395400                                                                  ValValGluGlyCysGlyCysArg                                                       405                                                                          (2) INFORMATION FOR SEQ ID NO:7:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                       SerAlaProGlyArgArgArgGlnGlnAlaArgAsnArgSerThrPro                               151015                                                                       AlaGlnAspVal                                                                  20                                                                            (2) INFORMATION FOR SEQ ID NO:8:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                      SerXaaLysHisXaaXaaGlnArgXaaArgLysLysAsnAsnAsn                                 151015                                                                        (2) INFORMATION FOR SEQ ID NO:9:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 amino acids                                                     (B) TYPE: amino acid                                                         (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                       SerThrGlyGlyLysGlnArgSerGlnAsnArgSerLysThrProLys                              151015                                                                        A snGlnGluAla                                                                 20                                                                            (2) INFORMATION FOR SEQ ID NO:10:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 11 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                      XaaValValLeuLysAsnTyrGlnAspMetVal                                              1510                                                                         (2) INFORMATION FOR SEQ ID NO:11:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 12 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                      XaaXaaLysValValLeuLysAsnTyrGlnAsp Met                                         1510                                                                          (2) INFORMATION FOR SEQ ID NO:12:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                                      SerAlaProGlyArgArgArgGlnGl nAlaArgAsnArgSerThrPro                             151015                                                                        AlaGlnAspVal                                                                  20                                                                            (2) INFORMATION FOR SEQ ID NO:13:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                                      AsnProGluTyrValProLysXaaXaaXaaAlaProThrLysLeuAsn                              151015                                                                        AlaIleS erVal                                                                 20                                                                            (2) INFORMATION FOR SEQ ID NO:14:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                                      XaaAlaThrAsnXaaAlaIleValGlnXaaLeuValXaaLeu Met                                151015                                                                        (2) INFORMATION FOR SEQ ID NO:15:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 6 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                                      XaaValXaaAla XaaGly                                                           15                                                                            (2) INFORMATION FOR SEQ ID NO:16:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 8 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                                      LeuTyrLeuAspGluAsnGluLys                                                      1 5                                                                           (2) INFORMATION FOR SEQ ID NO:17:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 8 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:                                      ValValGluGlyXaaGlyXaaArg                                                      15                                                                            (2) INFORMATION FOR SEQ ID NO:18:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 25 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:                                      LeuTyrLeuAspGluAsnGluLysValValLeuLysAsnTyrGlnAsp                              151 015                                                                       MetValValGluGlyXaaGlyXaaArg                                                   2025                                                                          (2) INFORMATION FOR SEQ ID NO:19:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:                                     SerThrGlyGlyLysGlnArgSerGlnAsnArgSerLysThrProLys                              151015                                                                        AsnGlnGluAla                                                                  20                                                                            (2 ) INFORMATION FOR SEQ ID NO:20:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:                                      XaaAlaThrAsnHisAlaIleValGlnThrLeuValHisPheIleAsn                              15 1015                                                                       XaaGluThrVal                                                                  20                                                                            (2) INFORMATION FOR SEQ ID NO:21:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:                                       LeuTyrLeuXaaGluTyrAspXaaValValLeuXaaAsnTyrGln                                151015                                                                        (2) INFORMATION FOR SEQ ID NO:22:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 10 amino acids                                                    (B) TYPE: amino acid                                                          (D ) TOPOLOGY: linear                                                         (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:                                      SerAlaXaaXaaHisXaaIleValGlnThr                                                1510                                                                          (2) INFORMATION FOR SEQ ID NO:23:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 11 amino acids                                                    (B) TYPE: amino acid                                                          ( D) TOPOLOGY: linear                                                         (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:                                      XaaAlaThrAsnXaaAlaIleValGlnThrLeu                                             1510                                                                          (2) INFORMATION FOR SEQ ID NO:24:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 11 amino acids                                                    (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                         (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:                                      LeuTyrLeuAspGluXaaGluXaaValValLeu                                             1510                                                                          (2) INFORMATION FOR SEQ ID NO:25:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 8 amino acids                                                     (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                         (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:                                      XaaXaaXaaGlyArgXaaArgGln                                                      15                                                                            (2) INFORMATION FOR SEQ ID NO:26:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 7 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:                                      XaaXaaGlyGlyXaaGlnArg                                                         15                                                                            (2) INFORMATION FOR SEQ ID NO:27:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 13 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:                                      LeuTyr LeuAspXaaAsnXaaXaaValValLeuXaaAsn                                      1510                                                                          (2) INFORMATION FOR SEQ ID NO:28:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 7 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:                                       XaaProGluXaaValProXaa                                                        15                                                                            (2) INFORMATION FOR SEQ ID NO:29:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:                                      SerAlaProGlyArgArgArg GlnGlnAlaArgAsnArgSerThrPro                             151015                                                                        AlaGlnAspVal                                                                  20                                                                            (2) INFORMATION FOR SEQ ID NO:30:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 amino acids                                                     (B) TYPE: amino acid                                                         (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:                                      SerThrGlyGlyLysArgArgSerGlnAsnArgSerLysThrProLys                              151015                                                                        As nGlnGluAla                                                                 20                                                                            (2) INFORMATION FOR SEQ ID NO:31:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 11 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:31:                                      SerThrGlyGlyLysGlnArgSerGlnAsnArg                                              1510                                                                         (2) INFORMATION FOR SEQ ID NO:32:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 32 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:32:                                      WSNACNGGNGGNAARCARMGNWSNCARAAY MG32                                           (2) INFORMATION FOR SEQ ID NO:33:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 13 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:33:                                      AsnHisAlaIleValGlnThrLeuValHisPhe IleAsn                                      1510                                                                          (2) INFORMATION FOR SEQ ID NO:34:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 53 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (ix) FEATURE:                                                                 (A) NAME/KEY: misc difference                                                  (B) LOCATION: replace (18, 27, 30, 33, 39, 42, 45)                           (D) OTHER INFORMATION: note="All 'N's in this sequence                        designate the nucleotide analog deoxyinosinetriphosphate                      (dITP) which was used in the positions where all four of                      the nucleotides (A, C, T or G) were possible."                                (xi) SEQUENCE DESCRIPTION: SEQ ID NO:34:                                      TTTTTTTTGGATCCRTTNATRAARTGNACNARNGTYTGNACNATNGCRTGR TT53                      (2) INFORMATION FOR SEQ ID NO:35:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 10 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:35:                                      LysThrProLysAsnGlnGluAlaLeuArg                                                15 10                                                                         (2) INFORMATION FOR SEQ ID NO:36:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 29 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (ix) FEATURE:                                                                 (A) NAME/KEY: misc difference                                                 (B) LOCATION: replace (3, 6, 9, 18, 24, 27)                                   (D) OTHER INFORMATION: note="All 'N's in this sequence                        designate the nucleotide analog deoxyinosinetriphosphate                      (dITP) which was used in the positions where all four of                      the nucleotides (A, C, T or G) were possible."                                (xi) SEQUENCE DESCRIPTION: SEQ ID NO:36:                                      AANACNCCNAARAAYCANGARGCNYTNMG29                                               (2) INFORMATION FOR SEQ ID NO:37:                                             ( i) SEQUENCE CHARACTERISTICS:                                                (A) LENGTH: 25 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:37:                                      GAGCAAGTTCAGCCTGGTTAAGTCC25                                                   (2) INFORMATION FOR SEQ ID NO:38:                                              (i) SEQUENCE CHARACTERISTICS:                                                (A) LENGTH: 25 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:38:                                      TGGCTTATGAGTATTTCTTCCAGGG25                                                   (2) INFORMATION FOR SEQ ID NO:39:                                              (i) SEQUENCE CHARACTERISTICS:                                                (A) LENGTH: 30 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:39:                                      GTCGCTGCTGCTGTTCTCTGCCACGTTGGC30                                              (2) INFORMATION FOR SEQ ID NO:40:                                              (i) SEQUENCE CHARACTERISTICS:                                                (A) LENGTH: 27 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:40:                                      GAATTCGTCGACATGCACGTGCGCTCA27                                                 (2) INFORMATION FOR SEQ ID NO:41:                                              (i) SEQUENCE CHARACTERISTICS:                                                (A) LENGTH: 22 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:41:                                      CCATGGCGTTGTACAGGTCCAG22                                                      (2) INFORMATION FOR SEQ ID NO:42:                                              (i) SEQUENCE CHARACTERISTICS:                                                (A) LENGTH: 9 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:42:                                      GlnAlaLysHisLysGlnArgLysArg                                                   15                                                                            (2) INFORMATION FOR SEQ ID NO:43:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 27 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:43:                                      CAAGCCAAACACAAACAGCGGAAACGC27                                                 (2) INFORMATION FOR SEQ ID NO:44:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A ) LENGTH: 37 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:44:                                      AAGCTTCCGCGGCTAGCGACACCCACAACCCTCCACA37                                       (2) INFORMATION FOR SEQ ID NO:45:                                             (i) SEQUENCE CHARACTERISTICS:                                                 ( A) LENGTH: 26 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:45:                                      ACTGTCGACATGGTGGCCGGGACCCG26                                                  (2) INFORMATION FOR SEQ ID NO:46:                                             (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 26 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:46:                                      ACGTTTTTCTCTTTTGTGGAGAGGAT26                                                  (2) INFORMATION FOR SEQ ID NO:47:                                             (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 21 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:47:                                      TGAAGCGGCCGCAACAGACGT21                                                       (2) INFORMATION FOR SEQ ID NO:48:                                             (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 21 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:48:                                      CTGTTGCGGCCGCTTCAACGT21                                                       (2) INFORMATION FOR SEQ ID NO:49:                                             (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 8 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:49:                                      MetIleProGlyAsnArgMetLeu                                                      15                                                                            (2) INFORMATION FOR SEQ ID NO:50:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 36 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:50:                                      GAATTCGTCGACATGATTCCTGGTACCGAATGCTGA36                                        (2) INFORMATION FOR SEQ ID NO:51:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 7 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:51:                                      ValGluGlyCysGlyCysArg                                                         15                                                                            (2) INFORMATION FOR SEQ ID NO:52:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 38 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D ) TOPOLOGY: linear                                                         (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:52:                                      AAGCTTCCGCGGCTCAGCGGCACCCACATCCCTCTACT38                                      (2) INFORMATION FOR SEQ ID NO:53:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 26 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      ( D) TOPOLOGY: linear                                                         (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:53:                                      ACTACCGCGGTAAATGAGTGCGACGG26                                                  (2) INFORMATION FOR SEQ ID NO:54:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                         (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:54:                                      CACTGCATTCTAGTTGTGGT20                                                        (2) INFORMATION FOR SEQ ID NO:55:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 27 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                         (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:55:                                      CAAGCCAAACACAAACAGCGGAAACGC27                                                 (2) INFORMATION FOR SEQ ID NO:56:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 37 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                         (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:56:                                      AAGCTTCCGCGGCTAGCGACACCCACAACCCTCCACA37                                       (2) INFORMATION FOR SEQ ID NO:57:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                         (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:57:                                      TCCACGGGGAGCAAACAGCGCA22                                                      (2) INFORMATION FOR SEQ ID NO:58:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 27 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                         (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:58:                                      CATACCGCGGAGCTAGTGGCAGCCACA27                                                 (2) INFORMATION FOR SEQ ID NO:59:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 37 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                         (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:59:                                      GAATTCGTCGACATGATTCCTGGTAACCGAATGCTGA37                                       (2) INFORMATION FOR SEQ ID NO:60:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 27 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                         (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:60:                                      ACGCTTGGCCCTCCGGCGTCGGGTCAA27                                                 (2) INFORMATION FOR SEQ ID NO:61:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                         (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:61:                                      TTTTTTCCAGTCTTTTGGACACCAGGTTGG30                                              (2) INFORMATION FOR SEQ ID NO:62:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 37 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                         (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:62:                                      AAGCTTCCGCGGCTAGCGACACCCACAACCCTCCACA37                                       (2) INFORMATION FOR SEQ ID NO:63:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 7 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:63:                                      ValGluGlyCysGlyCysArg                                                         15                                                                        

What is claimed is:
 1. An osteogenic protein preparation comprising aheterodimer of a first polypeptide subunit and a second polypeptidesubunit the preparation produced according to the method of culturing ina suitable culture medium a cell line transformed with a first and asecond nucleotide sequence, said first nucleotide sequence beingselected from the group consisting of:the nucleotide sequence as shownin SEQ ID NO: 3; and a nucleotide sequence which encodes the samesequence of amino acids as encoded by the nucleotide sequence shown inSEQ ID NO: 3; and said second nucleotide sequence being selected fromthe group consisting of: the nucleotide sequence as shown in SEQ ID NO:1; and a nucleotide sequence which encodes the same sequence of aminoacids as encoded by the nucleotide sequence shown in SEQ ID NO: 1; toproduce said heterodimer, and isolating said preparation from theculture medium.
 2. A pharmaceutical composition consisting of anosteogenic protein preparation according to claim
 1. 3. A method forinducing bone formation in a mammal comprising administering to saidmammal an effective amount of the osteogenic preparation of claim
 1. 4.The method of claim 3 wherein said osteogenic preparation is admixedwith a physiologically acceptable matrix material, carrier or diluent.5. A composition for implantation into a mammal consisting of theosteogenic preparation of claim 1 admixed with a physiologicallyacceptable matrix material, carrier or diluent.
 6. The compositionaccording to claim 5 wherein said physiologically acceptable matrixmaterial is selected from the group consisting of tricalcium phosphate,hydroxyapatite, collagen, plaster of paris, thermoplastic resins,polylactic acid, polyglycolic acid and polycaprolactic acid.